Biomembrane Transport

Biomembrane

Transport

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Biomembrane Transport Lon J. Van Winkle Midwestern University

With contributions by Ovidio Bussolati, Gian Gazzola, and John McGiven Bryan Mackenzie, Milton H. Saier, Jr., Peter M. Taylor, Michael J. Rennie, and Sylvia Y. Low

A C A D E M I C PRESS San Diego

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This book is printed on acid-free paper. @ Copyright 9 1999 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Academic Press a division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www, apnet, com Academic Press 24-28 Oval Road, London NW1 7DX, UK http ://www.hbuk.co.uk/ap/ Library of Congress Catalog Card Number: 98-89087 International Standard Book Number: 0-12-714510-9 PRINTED IN THE UNITED STATES OF AMERICA 99 00 01 02 03 04 EB 9 8 7 6

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To my wife, Mikki, who taught me to learn by resolving differences.

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Contents

W. The Gibbs-Donnan Effect Also Generates

Foreword xi Preface xiii

Osmotic Pressure 47 VI. Chemical Reactions Drive Primary Active Transport 49 VII. Reversal of Transport May Drive Chemical Reactions 55 VIII. How Do Fluctuations in the Local Hydrogen Ion Potential Facilitate Formation of Phosphoric Acid Anhydride Bonds by the Mitochondrial FoFI_IATP Synthase? 56 IX. Conversion of Solute Total Chemical Potential Gradients to Gradients of Other Solutes during Co- and Countertransport 57 X. Dissipation of Solute Gradients through Mediated Transport Processes May Also Perform Work 61 XI. Application of Thermodynamic Principles to the Solution of Practical Transport Problems 63 XII. Summary 63

1. I m p o r t a n c e of B i o m e m b r a n e T r a n s p o r t I. Introduction 1 II. Solute and Solvent Fluxes Are Determined by Barriers and Propelling Forces 3 III. Biomembrane Transport in Context 7 IV. Summary 10

2. Biomembrane Composition, S t r u c t u r e , and Turnover I. Introduction 13 II. Is the Fluid Mosaic Model of Membrane Structure Still Adequate? 13 III. Some Components of the Biomembrane Can Be Reconstituted 29 IV. How Are Biomembrane Composition and Structure Regulated? 30 V. Summary 38

4. T r a n s p o r t Kinetics I. Introduction 65 II. Kinetics of Diffusion 66 III. How Do Measurements of both the Diffusional and the Osmotic Permeability Coefficient for Water Inform Us about the Mechanism of Water Transport across a Plasma Membrane? 70 IV. Do Lipophilic Substances Migrate across Biomembrane Phospholipid Bilayers by Simple Diffusion? 73 W. Lipid-Soluble Substances Are Used to Attempt to Measure the Width of Unstirred Water Layers on Either Side of Biomembranes 74

3. T h e r m o d y n a m i c s a n d T r a n s p o r t I. Introduction 39 II. Similar Mathematical Expressions Serve for the Free Energy Change in a Chemical Reaction and in the Migration of a Solute or Solvent 39 III. Changes in Enthalpy and Entropy May Contribute Differently to the Free Energy Changes Associated with a Biochemical Reaction and Migration of a Solute 43 IV. The Total Chemical Potential Change for a Transport Process Also May Have an Electrical Component 44

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VI. Do Such Determinations of the Apparent Widths of Unstirred Water Layers Reflect the Intended Physical Phenomenon or Our Ignorance of How Lipid-Soluble Substances Cross Biomembranes? 76 VII. Protein versus Lipid-Mediated Mechanisms of Fatty Acid Migration across Biomembranes 79 VIII. Protein-Mediated Biomembrane Transport Is Probably Always Substrate Saturable 81 IX. Kinetics of Saturable Transport 83 X. Identification and Minimization or Deduction of Processes That May Obscure a Transport Process of Interest 98 XI. Kinetic Differences among Substrate-Saturable Transport Processes That Form, Propagate, or Dissipate Solute Gradients 116 XII. Summary 124 Appendix 126 5. Structure a n d Function of Transport Proteins That Form Solute Gradients I. II. III. IV.

Introduction 133 P-Type ATPases 135 FoFI-ATP Synthases (F-Type ATPases) Summary 166

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6. Transport Proteins That P r o p a g a t e Solute Gradients

II. III.

IV. V~

Introduction to Symporters and Antiporters 169 Both Erythroid and Nonerythroid Tissues Express Anion Exchangers 170 ASC and Excitatory (Anionic) Amino Acid Transporters Comprise One of Two Known Families of Mammalian Na+/Amino Acid Symporters 208 Both AE and EAAT/ASC Proteins Have Additional Functions 233 Summary 237

8. A P r o p o s e d S y s t e m for t h e Classification of T r a n s m e m b r a n e Transport Proteins in Living O r g a n i s m s Io Introduction 265 II. Work of the Enzyme Commission as a Basis for the Systematic Classification of Transport Proteins 265 III. Phylogeny as a Basis for Protein Classification: Criteria for Family Assignment 266 IV. Proposed Transport Protein Classification System 267 go Representative Examples of Classified Families 272 VI. Cross-Classification of Transport Proteins 272 VII. The Two Largest Superfamilies of Transporters: The MF and ABC Superfamilies 275 VIII. Macromolecular Transport Proteins in Bacteria 275 IX. Conclusions and Perspectives 276

9. Regulation of Plasma M e m b r a n e Transport I~ Introduction 277 II. Regulation of Transport by Changes in Driving Force: The Role of Plasma Membrane Potential 277 III. Regulation of the Activity of Existing Transporters through Modifications of Transporter Molecules 278 IV. Regulation of Transport by Changes in the Repertoire of Transport Proteins in the Plasma Membrane 284 V~ Coordinated Regulation of Transport Systems 287 VI. Derangements in Transport Regulation 287 VII. Summary 293

10. B i o m e m b r a n e Transport a n d I n t e r o r g a n Nutrient Flows: The A m i n o Acids I~ Interorgan Nutrition

II. 7. Channel Proteins Usually Dissipate Solute Gradients I. Introduction 239 II. Structure, Function, and Evolution of Channel Proteins 240 III. Kinetics of Transport via K + and Other Channels 254 IV. Summary 262

III. IV. V~

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295 Interorgan Amino Acid Nutrition: General Principles and Key Issues 295 Control of Interorgan Amino Acid Metabolism: Metabolic Control Theory and Safety Factors 308 Physiologically Important Flows of Amino Acids and Related Compounds 311 Amino Acid Nutrition under Special Circumstances 319 Summary 325

Contents

1 1. S e l e c t e d T e c h n i q u e s in M e m b r a n e Transport I. Introduction 327 II. Purification and Reconstitution of Transport Proteins 327 III. Methods for Isolating cDNAs Coding for Transport Proteins 328 IV. Heterologous Expression Systems for Transport Proteins 329 V. Voltage-Clamp Techniques in Xenopus Oocytes 332 VI. Probing Transport with Ion-Selective Microelectrodes 338

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VII. Optical Methods for Measuring Membrane Transport 339 VIII. Structure-Function Studies of Transport Proteins 339 IX. Genetic Approaches to Understanding Transporter Function 341 X. Summary of Preparations Used to Study Native Membrane Transport 341 XI. Commentary Epilogue 343 References 345 Index 387

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Foreword

Originally conceived as an update and expansion of my 1975 edition of Biological Transport, Van Winkle's Biomembrane Transport integrates recent advances in this broad field with several historically important concepts. Van Winkle argues convincingly that each of the transport proteins functions by interacting intimately with its specific substrate to provide a pathway for the movement of the substrate across a biomembrane. He points out that all such proteins need to move in order to catalyze transport, although the extent of the conformational changes varies greatly among the proteins. This perspective departs significantly from the view that the proteins that form, propagate, and dissipate solute gradients across biomembranes function by a variety of distinct mechanisms. My good impression of Van Winkle's efforts at this integration of transport is strongly heightened by his attention to detail. In separate chapters of this book, Dr. Van Winkle describes what is known about the structures and catalytic mechanisms of several examples of each category of transport protein. In this process he exposes differences as well as similarities in the structures and mechanisms of action of proteins of the same and different categories. What stands out in each of these chapters is how frequently the actual thermodynamics and kinetics of substrate transport appear to differ from currently accepted formulations for the transport. These revelations add up to an important contribution to a field in which numerous investigators are pressing to discover details of transporter structure and action, even though the characteristics of transport itself may still remain inadequately described and appreciated. With these caveats in mind, several guest authors integrate the actions of various types of transport pro-

teins in chapters on transporter regulation and the resulting interorgan flows of their substrates. I call attention especially to the remarkable, current development of the subject of competition of amino acids for transport across the blood-brain barrier presented in Chapter 10, particularly in phenylketonuria, where phenylalanine in excess is the dangerous competitor, and in maple syrup disease, where it is instead leucine, a leucinosis. Learning of such physiological and pathophysiological functioning of the transporters is of course the purpose for studying them, although this goal may sometimes be obscured in experiments using powerful new molecular procedures. In a guest chapter on some of these techniques, Dr. Bryan Mackenzie makes the important observation that we are likely to return to greater use of conventional preparations to study biomembrane transport as an appropriate emphasis of their overall physiology is restored. This concern urges the modern investigator to understand and be prepared to use a wide array of procedures available for studying transport at the subcellular, cellular, tissue, and organismal levels of biological organization. Additionally, we can comprehend fully the breadth of our field of biomembrane transport by examining carefully for its bounds, for example, among enzymes whose characteristic actions differ from those of transporters in destabilizing their substrates rather than in simply moving them from one phase to another. In short, Lon Van Winkle's effort helps us very much in describing what we know and what we do not know about biomembrane transport. I believe he has done an outstanding job. He has ranged thoughtfully in his invitation of guest authors to broaden his already good perspective. His book asks many questions and provides good answers. For myself, a person who has faced questions on membrane transport for about a half-century,

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I find this book both insightful and provocative. Watching it evolve has been very satisfying and rewarding to me, I having until now privately included Lon Van Winkle among my own students, even though no such formal arrangement was ever made. I encourage other stu-

dents of membrane transport to study this book to seek the benefits of the continued development of the field.

Halvor Christensen

Preface

Some new investigators may find themselves in the field of biomembrane transport in part serendipitiously because an interesting cDNA clone happened to encode a transport protein. Others may have been led to the field through their investigations relatively late in their careers, well after their formal training was complete. It is hoped that this book will help such individuals fill deficiencies that may exist in their knowledge of biomembrane transport. Beyond this more limited goal, the book is intended to give any interested student of biochemistry and molecular biology insight into what is as well as what is not yet known about biomembrane transport and its importance to the physiological functions of cells. The book is divided into three main parts. The first part (Chapters 2 to 4) covers fundamental principles of biomembrane structure and transport. In the second part (Chapters 5 to 7) we discuss the structures and functions of transport proteins that form, propagate, and dissipate solute total chemical potential gradients. Finally, three chapters (8 to 10), written by prominent guest authors, span the topics of classification, regulation, and integration of the functions of biomembrane transport proteins. Modern techniques for the study of biomembrane transport are discussed briefly in several sections of various chapters and in Chapter 11. Chapters 8 to 11 add not only important dimensions to the book, but also the unique perspectives of the guest authors. I leave it to the guest authors themselves to reveal their sometimes novel views on transport in their individual chapters and do not speak for them in this preface except coincidentally. Transport proteins have evolved on numerous occasions to catalyze migration of a solute or the solvent across biomembranes. Such evolution has been necessary because membrane lipid bilayers otherwise present virtually impenetrable barriers to most hydrophilic sol-

utes. Hence, it became possible to regulate the composition of intracellular and extracellular fluids with the advent of biomembrane transport proteins. Moreover, such regulation was made progressively more sophisticated as more types of proteins evolved to transport the same as well as different solute species. Modern organisms appear now to need such diversity of biomembrane transport processes to compete successfully with other species. Such circumstances also mean, however, that the biomembrane transport proteins that evolved in apparently unrelated families and superfamilies nevertheless evolved under similar constraints; new transport processes have had to improve the ability of the organism to fit into a successful niche in the biological community by influencing a single main function of their cell or cells. Consequently, virtually all such biomembrane transport proteins function in two fundamentally similar ways. 1 First, they provide pathways for the migration of their substrates across biomembranes. Such pathways involve temporary association of the substrate with one or more sites along the pathway, thus rendering the pathways selective for one or a few chemically and physically similar solutes. Moreover, such mediated transport is substrate saturable apparently because the interactions between substrate and transport protein necessarily slow migration of the solute relative to the rate at which it could migrate over the same distance by ordinary diffusion. Nevertheless, the rate of biomembrane transport varies among proteins over nearly 10 orders of magnitude, apparently owing to a need for 1 We are discussing here the majority of transport proteins that are produced by organisms for their own uses. Not included in this summary are transport proteins, such as cz-hemolysin, that are produced by an organism in order to cause the death of the cells of another. The latter proteins function by insertion of the transport protein molecules into the plasma membranes of cells of the target organism.

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such differences in rate under various physiological conditions. While it is conceivable that differences in the way in which transport proteins interact with their substrates could alone account for their wide range of known transport rates, we think this wide range in rates also depends on differences in the magnitude of the other fundamentally similar way in which virtually all transport proteins function. 2 As we shall see, virtually all biomembrane transport proteins need to move in order to catalyze transport. In the cases of transport proteins historically known as carriers or primary active transporters, such conformational changes may be relatively large and easy to document, although none of the proteins completely reverses its conformation across the membrane. In contrast, the more rapid migration of solutes and the solvent across the membrane via channels appears to require only the small movements that all macromolecules normally undergo. Some readers may question whether we have accepted prematurely data showing that channel proteins must be able to move normally in order to catalyze transport. These data are based primarily on computer simulations of protein structure and on similarities between channel proteins and enzymes in their interactions with substrates. We maintain, however, that the principal reason the movements of channel proteins during transport are not as well documented as the movements of other types of transport proteins is that channel proteins are not expected to move except to open, close, or inactivate. However, as for all proteins and other molecules at temperatures well above absolute zero, channel proteins and other membrane constituents do exhibit predictable motion, and their movements as well as that of the substrate are needed for transport to occur.

In a similar vein, we challenge the common notion that transport in some cases occurs by a process that resembles ordinary diffusion. The notion of transport by diffusion of course contradicts the theory that transport via transmembrane pathways formed by proteins requires the proteins to move during transport. As we shall see, however, it is also our position that even lipophilic solutes do not appear to migrate across the highly ordered lipid bilayers of biomembranes by processes that resemble ordinary diffusion. If we are right, one consequence would be that the widths of the unstirred 2 Use of the word "we" to refer to the primary author here or in other sections of this book should not be taken to mean that guest authors share all of his opinions about biomembrane transport. The opinions expressed in each chapter are those of the author or authors of it and may or may not be shared by the others.

water layers on either side of the lipid bilayer have been vastly overestimated. We hope that readers will accept our good intention of such challenges to common theories and beliefs about the mechanisms of biomembrane transport. We accept at the outset that many of our notions may be incorrect, but we think that accepted paradigms may themselves also not be well supported by experimental data. Our purpose then is to provoke thought and further study in these instances. It is after all such an inquisitive spirit, as well as our disagreements, that inspires us to develop and test creative new theories about the functions of biomembrane transport proteins. The field of biomembrane transport also has become too broad for a detailed discussion of all important instances of such transport. Consequently, we discuss many principles that are pertinent to all transport processes, but the examples selected to illustrate these principles are only a very few of the numerous wellstudied examples that could have been chosen. Similarly, to discuss the relationship of protein structure to function in enough detail to present a full view of the state of the art, only some of the many important examples of transport proteins had to be selected. If, however, we are correct in our assertion that virtually all transport proteins function according to fundamentally similar principles and mechanisms, then selection of these examples should indeed give the reader the necessary insight into the broad field of biomembrane transport. Many people contributed to the production of this book, and I will not attempt to mention each one by name lest I forget someone more deserving than those I remember. Most people who helped to prepare the book are members of various departments at Midwestern University, including Biochemistry, Library Services, Media Resources, and Research Affairs. Individuals in these departments who must be mentioned by name because of the quantity of work they performed include Allan Campione, Barbara Le Breton, Michael Moore, and Eileen Suarez. Moreover, several colleagues provided constructive criticisms of more than one chapter, sometimes exposing differences in our opinions. Although many of these differences were constructively resolved, some still remain, so my colleagues are not to blame for my ideas about transport that may turn out to be wrong. Readers should also see the acknowledgments in individual guest chapters for persons who contributed to production of those parts of the book. Colleagues who reviewed several or all of the first seven chapters include Stefan Br6er, Halvor Christensen, Jacquelyn Smith, Susan Viselli, Douglas Webster, and James Young.

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1 Importance of Biomembrane Transport

1. INTRODUCTION Asexual, sexual, and cellular reproduction and the functioning of cells and organisms may be viewed in relation to various modifications of the central dogma. The dogma historically places nucleic acids and in particular D N A at a central position in biology. D N A is of course needed for organisms to reproduce and for them to pass their genes to the next generation. The only measure of an organism's biological productivity is the relative quantity of heritable D N A that it, and in some cases, its relatives contribute to subsequent generations. Despite the importance of nucleic acids to our comprehension of how living things function, other cellular constituents are of course required for cells and organisms to reproduce and remain alive. In particular, biocatalysts are needed both to interpret the information in nucleic acids and to propagate the cells and organisms that contain them (Fig. 1.1). Biocatalysts also convert free energy into biochemical and biophysical forms useful in performing the work of living and reproducing. Any biological molecule or combination of such molecules that increases the rate of a process in vivo qualifies here as a biocatalyst. Familiar forms of biocatalysts include enzymes, ribozymes, chaperones, and biomembrane transport proteins. The same biocatalyst molecule may also increase the rate of more than one process, as we will come to expect in this volume when we consider the multiple functions of many biomembrane transport proteins (especially in Chapter 6). Moreover, these multiple processes may be of the same type, such as multiple independent biomembrane transport processes, or they may be of different types, such as a transport process

that is coupled to a chemical change. For example, the F-type ATPases (or ATP synthases) of chloroplasts convert the free energy of the proton gradient formed by light-driven active transporters into the free energy normally realized in ATP when the ATPases also catalyze transport of protons along their total chemical potential gradient. Biocatalysts that function in biomembrane transport constitute a quantitatively significant portion of all proteins. As pointed out in Chapter 8 of this volume, recent complete genome analysis revealed that about 10% of all genes in microorganisms encode transport proteins. Moreover, catalysts are needed to insert these transport proteins asymmetrically into biomembranes. In the case of photosynthesis, proton gradients can be formed only if transport is asymmetric, and subsequent use of the gradients for ATP synthesis requires that the ATP synthases also function asymmetrically. As for the membranes in chloroplasts and other intracellular organelles, the plasma membrane also is asymmetric, and this asymmetric structure helps to organize important biological processes. For example, watersoluble signaling molecules bind to receptors on the outside of cells. As a result of such binding, a cascade of events often is produced within cells to change their metabolism (Fig. 1.2). These changes frequently involve net transport of solutes asymmetrically in one direction or the other across biomembranes. Clearly, the symmetric functioning of such a system would be of little value to cells. Hence, the asymmetry of the barriers that biomembranes form is as critical to their normal functioning as the transport proteins and other catalysts that are associated with them. The importance of this asymmetry to the normal functioning of the transport biocatalysts may some-

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1. Importance of Biomembrane Transport

FIGURE 1.1 Extension of the central dogma of molecular biology to include reverse transcription and RNA replication. Each of the processes depicted is needed by some or all organisms in order for them to function and survive. Solid arrows in the dogma and its extensions are meant to indicate the directions of information transfer. In addition, proteins and ribozymes are shown as containing information needed to catalyze the processes. Hence, the information needed to sustain life is contained both in nucleic acids and in biocatalysts.

times be more subtle than in the conspicuous instances just discussed. For example, the asymmetric functioning of inwardly rectifying K + channels appears to allow the channels to transport K + ions into cells against their total chemical potential gradient. As we shall see in Chapter 7, this asymmetric functioning depends primarily on the underlying asymmetry of a polyamine gradient with which the channels interact. If verified, this surprising transport would occur critically at the resting membrane electrical potential. Consequently, it makes the membrane more sensitive to depolarizing stimuli. The existence of K + channels in membranes was postulated in the first place because this ion appeared to traverse the hydrophobic interior regions of biomembranes more rapidly than anticipated from the hydrophilic character of K +. In the absence of a protein mediator, the rates at which many solutes permeate biomembranes appear to depend on their molecular masses and lipid solubilities. More hydrophobic substances are sometimes viewed as being able to permeate the phospholipid bilayers of biomembranes more easily than hydrophilic ones, owing in part to their ability to dissolve in and subsequently diffuse through the hydrophobic region at the center of such bilayers. Diffusion of smaller solutes is of course more rapid than larger

FIGURE 1.2 Binding of hydrophilic signaling molecules to their receptors on the outside of a cell frequently activates a cascade of events in the plasma membrane and cytosol. In the case depicted, the norepinephrine-bound receptor actually can stimulate numerous Gprotein molecules (shown as a single o~-subunit that has separated from the 3'- and/3-subunits) each to activate an adenylate cyclase molecule. One result of the signaling in this case is the asymmetric net transport of Ca 2+ into cells along its total chemical potential gradient. The whole system must, of course, also operate asymmetrically across the membrane to be effective (adapted from Opie, 1991, with permission from Lippincott-Raven Publishers).

ones, so better correlations between permeability and hydrophobicity are obtained when the permeabilities are corrected for the size of the solute. Hence, when a substance appears to permeate a biomembrane more rapidly than anticipated from these properties, it becomes reasonable to look for a transport process that may mediate migration of the solute across the membrane. For example, the paradoxically very rapid transport of the solvent water across biomembranes may now be understood largely owing to the presence of water channel proteins in the membrane (see Sections II and III of Chapter 4 for further discussion). Moreover, other membrane proteins, such as the Na+-dependent glucose transporter (Loike et al., 1996; Loo et al., 1996), appear to catalyze transport of significant amounts of water in addition to that catalyzed by water-specific channels. Nevertheless, the migration of water across artificial phospholipid bilayers is still, in our view, paradoxically rapid, and special ways of accommodating water molecules in the bilayer structure have been proposed to account for this migration (e.g., Haines, 1994).

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Solute and Solvent Fluxes

Similarly, other substances may pass across biomembranes more rapidly than anticipated from their molecular size and structure. As for such migration of water, the migration of these solutes across the phospholipid bilayer may be catalyzed by proteins. Alternatively, the solutes may migrate more rapidly because of asyet poorly appreciated properties that appear to permit more rapid permeation of the lipid bilayer than anticipated from molecular size and hydrophobicity alone. For example, c~-tocopherol is a highly lipid-soluble substance whose membrane permeability can be increased by converting it to the larger and less lipid soluble substance tocopherol succinate (Bonina et al., 1996). Hence, protein-mediated transport may not always be present when migration of a solute is more rapid than anticipated. 1 Conversely, protein-mediated transport cannot always be ruled out solely because a solute migrates across the membrane at a rate anticipated from its physical properties alone. Nevertheless, the ability of any molecular or ionic species to move across a biomembrane depends only on the degree to which the membrane serves as barrier to that migration.

evolved partially to overcome the barriers. 3 Rather than using cellular free energy to make biomembrane transport faster than the rate of migration that could be achieved by ordinary diffusion, this free energy is used instead in combination with biomembrane barriers to produce total chemical potential gradients of solutes across biomembranes. Transport along these gradients then serves to perform additional work such as ATP synthesis, signal transduction, and regulation of cellular volume. A. Unidirectional Solute or Solvent Flux D e p e n d s on the D e g r e e to Which a B i o m e m b r a n e Serves as a Barrier to That Migration

The unidirectional flux of a solute or the solvent across a biomembrane proceeds much more slowly than could occur if free diffusion were possible over the same distance. Even the fastest transport via channels has been estimated to proceed no more rapidly than about 8% of the rate that could be achieved owing to free diffusion (calculated by Stein, 1986; p. 202). 2 While it is an interesting theoretical question whether a system could be constructed to catalyze biomembrane transport at a rate exceeding that which would occur if ordinary diffusion were possible, it is difficult to imagine a need for such a system except perhaps in the case of macromolecules. Consequently, the barrier functions of biomembranes can be seen to be at least as important to the lives of cells as the transport processes that have

In transport that is not saturable by substrate, the rate at which the substrate traverses the membrane depends only on the total chemical potential of the substrate, the total surface area of the membrane, and the permeability of the membrane to the substrate. The rate of nonsaturable unidirectional transport is not usually coupled to an obvious source of cellular free energy, nor does it depend on the concentration of the substrate on the other side of the membrane. For example, the unidirectional flux of a solute at a concentration of, say, 1.0 mM will occur at the same rate regardless of whether the solute concentration on the other side of the membrane is 0.1 or 10 mM. The rate of protein-mediated, substrate-saturable transport also need not be influenced by the concentration of the same substrate on the other side of the membrane, although unidirectional flux in the reverse direction will, of course, depend on this concentration. When the rate of mediated unidirectional transport is not influenced by the presence of the same substance or ion on the other side of the membrane, the transport is believed to be catalyzed by uniporters. Such transport is also sometimes imprecisely attributed to facilitated diffusion of the solute across the membrane via a carrier as discussed further in Section VIII of Chapter 4. As for biomembrane barriers, propelling forces influence protein-mediated unidirectional solute and solvent fluxes. The simplest of these forces is the total chemical potential gradient of the substrate. While a mathemati-

1Undetected mediation of transport by a protein is, of course, nearly impossible to rule out formally for any biomembrane. 2A possible exception to the results of this calculation may be transport via nonselective channels formed by some toxins such as a-hemolysin. These toxins form relatively wide pathways for the migration of water and most solutes. To our knowledge, the transport rates via these toxin channels has not been determined and compared to the rate that could occur if ordinary diffusion were possible.

3Similarly, other transport processes increase the rate of migration of inorganic and organic solutes in the cytosol by helping the solutes partially to overcome barriers to their free diffusion (e.g., Bronner, 1996; Luxon, 1996; Weisiger, 1996). However, the transport processes do n o t help the solutes to exceed their rates of ordinary diffusion. Terms, such as "self-diffusion", that are sometimes applied to the uncatalyzed migration of solutes in the cytosol are not equivalent to free diffusion of the solutes in the absence of cytosolic barriers.

II. SOLUTE AND SOLVENT FLUXES ARE DETERMINED BY BARRIERS AND PROPELLING FORCES

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1. Importance of Biomembrane Transport

cal expression for this gradient will be derived in Chapter 3, the reader's existing concept of total chemical potential should more than suffice for the present discussion. B. A Propelling Force is N e e d e d to P r o d u c e N e t Flux of a Solute or t h e Solvent in O n e Direction across a B i o m e m b r a n e Since propelling forces influence unidirectional flux, they also produce net flux when the propelling force is greater in one direction across the membrane than in the other direction. In the simplest case, a substance or ion migrates more rapidly along its total chemical potential gradient toward thermodynamic equilibrium than it moves in the reverse direction. As we discuss in several chapters, however (e.g., Chapters 4 to 7), this simple phenomenon does not account fully for the net transport catalyzed by most proteins. An exception appears to be the transport catalyzed by proteins, such as c~-hemolysin, that do not select among low-molecularweight solutes. All transport proteins that are substrate selective and saturable can be made to catalyze transport of a substrate against a total chemical potential gradient by coupling the transport to another source of free energy. C. The E n d e r g o n i c Migration of a Solute a g a i n s t Its Total Chemical Potential G r a d i e n t Can O c c u r O n l y W h e n It Is C o u p l e d to an E x e r g o n i c Process of G r e a t e r M a g n i t u d e Even uniporters (Section XI,G of Chapter 4) and channels (Section II,C of Chapter 7) may in some cases use the total chemical potential gradient of one substrate (or inhibitor) to generate a gradient of another. Such interconversions of gradients by uniporters and channels are, however, usually much less conspicuous and efficient than the propagation of one solute gradient into that of another by symporters and antiporters. The latter proteins couple the migration of one or more solutes to the co- or countermigration, respectively, of one or more other solutes. Hence, they are sometimes also termed cotransporters, countertransporters, exchange-transporters, or even secondary active transporters. The tightness of the coupling (i.e., the degree to which transport occurs only in the presence of all co- or countersubstrates) helps to determine how efficiently the free energy of one solute gradient is converted into that of another. When slippage or tunneling (i.e., uncoupled transport) is relatively frequent, the free energy transfer is relatively inefficient, whereas tightly coupled transport results

in the nearly complete conversion of the free energy in the gradient of one solute into that of another. Interestingly, transport in which coupling between co- (or counter-) substrates is not obligatory could lead to transport of a large amount of one cosubstrate relative to the other. This high ratio could be mistaken for the stoichiometry of comigration rather than the stoichiometry of cotransport of the substrates. 4 Such a high apparent stoichiometry of comigration of one substrate relative to the other would, however, actually reflect a high degree of uncoupling rather than the actual stoichiometry of comigration. Consequently, a total chemical potential gradient of the cosubstrate transported in greater amount would be dissipated without much transport of the other cosubstrate against its gradient. In contrast, the stoichiometry of cotransport of one cosubstrate relative to the other may be high, owing to the obligatory comigration of several ions or molecules of this first substrate to the transport of a single ion or molecule of a second kind. In this case, a total chemical potential gradient of the first cosubstrate across a membrane could produce a much steeper gradient of the second one, assuming only that a mechanism exists to maintain a steady-state gradient of the first cosubstrate (to be discussed further in Section IX,B of Chapter 3). Many solute gradients are maintained across biomembranes by coupling them to yet another source of free energy such as that realized from A T P hydrolysis. Conversely, transport along these gradients may drive A T P synthesis. When transport along a gradient normally coupled to A T P synthesis occurs without such coupling, however, additional free energy must be expended to maintain the gradient. In fact, when thermodynamically coupled processes are uncoupled for the purpose of generating thermal energy in mammals, uncoupled transport rather than uncoupled A T P hydrolysis results in thermogenesis. Rapid hydrolysis of A T P for thermogenesis might put at risk the numerous other cellular processes that rely on a well regulated A T P 4We define the stoichiometry of co- (or counter-) transport as the ratio of the number of ions or molecules of one substrate actually transported with a particular number of ions or molecules of the other substrate in the average transport cycle. This definition is contrasted here with our definition of the stoichiometry of co- (or counter-) migration, which is the number of ions or molecules of each species of substrate that are transported together in a single catalytic cycle of a transport protein. In the case of nonobligatory symport or antiport, the stoichiometry of comigration cannot be measured in every transport cycle since comigration does not occur in every cycle. Consequently, the stoichiometryof comigration may be difficult to determine experimentally, whereas the stoichiometry of cotransport can almost always be determined. Similarly, the stoichiometry of comigration may be difficult to determine experimentallywhen obligatory symport and antiport occur together, and different species of substrate have different probabilities of dissociating from the transport protein during its cycle (see Section III,B of Chapter 6 for further discussion).

Solute and Solvent Fluxes

supply. These processes are distributed throughout the cell, so the total volume of cytoplasm in which the ATP supply must be regulated is relatively large. In contrast, the ATP synthesis that is driven by a proton gradient in animals is restricted to the inner mitochondrial membrane. D. The Thermal Energy Released to Maintain a Solute Total Chemical Potential Gradient Provides Conspicuous Evidence of the Free Energy Content of the Gradient It is well established that an F-type ATPase catalyzes conversion of the free energy in the proton gradient across the inner mitochondrial membrane into the free energy realized in the phosphoric acid anhydride bonds of ATP (Chapter 5). The considerable free energy associated with this proton gradient becomes even more conspicuous in mammals when the proton gradient is disconnected from ATP synthesis by mitochondrial uncoupling proteins (UCPs). The thermal energy that is generated in opposing the action of UCPs serves both to warm the animal under cold stress and as a device to rid the animal of excess dietary free energy intake. While the first of these proteins to be discovered (UCP1) is expressed exclusively in mitochondria of brown adipose tissue (Ricquier et al., 1991), a second protein (UCP2) is widely distributed in the tissues of mammals including humans (Harper, 1997; Wolf, 1997). Twenty to 40% of mammalian mitochondrial oxygen consumption is needed to support the proton transport catalyzed by proteins such as UCP2 (Harper, 1997; Rolf and Brown, 1997). UCPs catalyze uncoupled H + transport across the inner mitochondrial membrane by a process that is distinct from the H + transport catalyzed by F-type ATPases. Hence, UCPs do not act to uncouple H + transport from ATP synthesis by F-type ATPases. Rather, they compete with F-type ATPases to transport protons and thus reduce the quantity of protons that could otherwise be used for ATP synthesis by 20 to 40%. The proton transport catalyzed by UCPs is associated with transport of a variety of inorganic and organic anions (Garlid, 1990), and physiologically important ones appear to be ionized fatty acids (Garlid et al., 1996; Jezek et al., 1997). Proton transport appears, however, not actually to be coupled to mediated fatty acid transport. Rather, UCPs are believed to catalyze uniport of fatty acids and other anions (Garlid, 1990). In the case of fatty acids, uniport of their anionic form out of mitochondria could be followed by their uncatalyzed migration into mitochondria in association with protons (Gar-

5

lid et aL, 1996; Jezek et aL, 1997). The latter migration of uncharged fatty acid molecules across the membrane may be relatively rapid, whereas transport of fatty acids in their normally anionic form appears always to be transport protein mediated (see Section VII of Chapter 4). Consequently, UCPs appear to catalyze uncoupled proton transport indirectly by enabling fatty acids to behave as cycling protonophores (Skulachev, 1991; Garlid et aL, 1996; Wojtczak et aL, 1998) (Fig. 1.3). The presence of possible proton-conducting groups on the side chains of some functionally important amino acid residues in UCP1 has, however, militated against universal acceptance of this protonophore theory (e.g., Bienengraeber et aL, 1998). Moreover, other investigators have concluded that fatty acids do not increase the rate of proton transport as a result of their own transport by UCPs (Gonzalez-Barroso et al., 1998). Regardless of the mechanism of proton transport owing to UCPs, the thermal energy generated in opposing the action of UCPs exposes the free energy content of proton gradients. Interestingly, UCPs are homologous to several other mitochondrial transport proteins including the ATp4-/ ADP 3- and H2POa-/OH- antiporters (Aquila et al., 1987; Klingenberg, 1990). Although the phosphate transporter was originally believed to catalyze H2PO4-/ H § cotransport, more recent evidence indicates that it

O

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Mitochondrial Matrix

FIGURE 1.3 Scheme showing how fatty acids may act as cycling protonophores to facilitate proton transport across the inner mitochondrial membrane. In this model it is proposed (Skulachev, 1991; Garlid et al., 1996) that uncoupling proteins (UCPs) catalyze uniport of fatty acid anions out of the mitochondrial matrix. The fatty acid anions are proposed, then, to associate with protons at the outer surface of the inner mitochondrial membrane, owing to the relatively high concentration of protons there. The undissociated fatty acids migrate relatively rapidly across the lipid bilayer without the help of a biocatalyst, whereas the fatty acid anions require a transport protein (in this case a UCP) to catalyze their migration. Once inside the mitochondrial matrix, the fatty acids dissociate from protons, owing to the relatively low proton concentration. While other authors (Skulachev, 1991; Garlid et al., 1996) show the UCP-catalyzed transport of the anionic forms of fatty acids as a "flippase" (to be discussed in Chapter 2), the actual mechanism by which these forms of fatty acids may migrate across the membrane via UCPs remains to be determined.

6

1. Importance of Biomembrane Transport

catalyzes H2PO4-/OH- exchange (Stappen and Kr~imer, 1994). Extrusion of O H - would of course accomplish the same end as H + uptake. Hence, although UCPs do not appear to catalyze H + transport directly, they could conceivably be modified effectively to do so. Such mutability of both substrate selectivity and the combinations of co- and countersubstrates received by transport proteins appears to have resulted frequently in the evolution of important new physiological functions in many families of such proteins (see the summary of families that contain homologous members in Chapter 8).

ies is found in the E A A T / A S C family of amino acid transporters (Chapter 6). Proteins in the E A A T subfamily catalyze the concentrative uptake of anionic amino acids in neurons and other tissues at the expense of both the Na+and K § gradients across the plasma membrane (Fig. 1.4). Consequently, many of these proteins help to reduce the glutamate concentration in the vicinity of glutamate receptors in the central nervous system to a level below the values of the dissociation constants of these receptors. Interestingly, however, several members of the E A A T subfamily appear to have evolved to express primarily a related but quite different additional function of the proteins. The latter members of the E A A T subfamily are postsynaptic proteins that catalyze mainly glutamate-stimulated C1- transport (Fig. 1.4) and relatively little glutamate transport (e.g., Fairman et al., 1995; Sonders and Amara, 1996; Arriza et al., 1997). Hence, some E A A T proteins may have a central rather than an auxiliary role in signal transduction (Sonders and Amara, 1996). Likewise, members of the ASC subfamily apparently evolved in yet another context to catalyze Na+-dependent exchange of zwitterionic amino acids (Fig. 1.4). Each of these different transport functions is important to the ability of different cells to perform their specialized functions. Another way in which changes in substrate selectivity may contribute to the evolution of important new functions among related proteins is for the stoichiometry of transport but not the substrate species themselves to change. We discuss in Chapter 5 the importance of

E. M e d i a t e d Transport Is Substrate Selective Biomembranes function as barriers to form compartments and consequently to organize metabolism among tissues and organs as well as among subcellular organelles. These functions of various biomembranes also depend, however, on the different substrate selectivities of their transport proteins. Hence, for example, the improper sorting of the homologous H+K +- and Na+K +selective ATPases to the basolateral and apical membranes, respectively, of an acid-secreting epithelium (instead of the other way around) would have disastrous consequences for the organism. In these cases, protons would be secreted inappropriately into interstitial spaces, whereas Na § would be extruded incorrectly into the lumens of pertinent organs such as the stomach (see also Section II,B,5 of Chapter 5). Another example of the importance of the evolution of transport proteins with different substrate selectivit-

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_

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FIGURE 1.4 Schemeto emphasize the various transport functions and relative substrate selectivities of different members of the EAAT/ASC protein family. The sizes of the abbreviations of the substrates are meant to indicate the relative amounts of transport by each group of transport proteins. EAAT1 to EAAT3 catalyze concentrative uptake of anionic amino acids such as glutamate (Glu-) to a greater extent than they catalyze glutamate-stimulated C1- transport as channels. In contrast, EAAT4 and EAAT5 catalyze more glutamate-stimulated C1- transport than they do glutamate uptake. Members of the other subfamilyof transport proteins in the EAAT/ASC family (i.e., the ASC subfamily) catalyze Na+-dependent exchange of zwitterionic amino acids (AA -~) as well as channel-like C1- transport. The relative amounts of these transport activities remain, however, to be determined for different ASC proteins. For this reason, they are shown approximately to be equal for ASC proteins, although such may not be the case for different members of this subfamily.

7

Biomembrane Transport in Context

V-type ATPases in acidification of intracellular compartments at the expense of ATP hydrolysis. On the other hand, ATP synthesis is usually accomplished in oxidative tissues by the related F-type ATPases in mitochondria. Part of the explanation of how these two homologous families of ATPases evolved to perform opposite functions is that the stoichiometry of H + ions transported per ATP molecule hydrolyzed or synthesized is lower by one or two protons in V-type than in F-type ATPases. For this reason, a much larger and usually unattained proton gradient would be required for V-type ATPases to carry out net ATP synthesis. Vtype ATPases may also catalyze some uncoupled proton transport in the reverse direction out of intracellular compartments (i.e., they may leak), which would help to make ATP hydrolysis by the enzyme irreversible. Similarly, F-type ATPases may also catalyze proton transport in either direction. Unlike V-type ATPases, however, proton transport remains coupled to ATP synthesis or hydrolysis in F-type ATPases. While reversal of the function of F-type ATPases is unusual in mitochondria, extrusion of protons at the expense of ATP hydrolysis under anaerobic conditions is a normal adaption of F-type ATPases is some bacteria. Hence, we see that differences in the reversibility of solute migration as well as in substrate selectivity combine with barrier action to determine a variety of functions of biomembranes in different cells and organelles. F. Reversibility of Solute Transport That solute transport must be reversible for optimum physiological functioning is no better exemplified than in the case of C1-/HCO3- exchange in the red blood cell (Chapter 6). The anion exchanger (AE1) catalyzes release of HCO3- from erythrocytes in exchange for C1in capillaries of respiring tissues (Fig. 1.5). The HCO3is produced in red blood cells by carbonic anhydrase, owing to their uptake of the CO2 produced in nearby cells. This process helps blood carry more total CO2 (CO2 plus HCO3-) than would otherwise be possible. A greater capacity for bulk flow of CO2 from peripheral tissues to the lungs appears to be particularly important during aerobic exercise. Anion exchange must be fully reversible, however, in order for erythrocytes to take up most efficiently HCO3- in exchange for C1- and convert it to CO2 for excretion by the lungs (Fig. 1.5). Similarly, we shall see that reversible solute transport into and out of cells is essential for normal nutrient flows among tissues and organs in other cases, such as in the fed and fasted states. These nutrient flows among tissues and organs will be discussed in Chapter 10 using amino acids as examples.

Lungsl

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FIGURE 1.5 Schemeto show why reversal of C1-/HCO3- exchange in erythrocytesis needed to help to carry CO2 from peripheral tissues to the lungs. Solid arrows show the net migration of the carbon in CO2 in the blood capillaries of peripheral tissues, whereas the dashed arrows show the net migration of the carbon within capillaries of lungs. Abbreviation: AE1, anion exchanger 1.

Transport may also be made reversible by using different transport processes to catalyze solute migration in one direction or the other across biomembranes. For example, Na+K+ATPase catalyzes K + uptake and Na + extrusion against their total chemical potential gradients across the plasma membrane of most animal cells. The reverse net transport of both cations is catalyzed by Na + and K + channels. In general, channels allow substrates to migrate along their total chemical potential gradients. Transport of Na + and K + in both directions across the membrane helps to produce and dissipate transmembrane electrical potentials in excitable cells, and it results in other types of cellular work, such as regulatory cellular volume increases and decreases. The activities of the transport processes themselves must also be regulated in these cases to produce physiologically desirable results (see Chapter 9 for further discussion of transport regulation).

III. BIOMEMBRANE TRANSPORT IN CONTEXT Most students of biomembrane transport eventually consider their findings in the broader context of the environments of cells in situ (e.g., see Chapter 10). Nevertheless, relatively few scientists actually study transport into the cells of perfused tissues, organs, and even whole multicellular organisms. Isolation and characterization of transport activities in a given cell type is particularly difficult in the latter context. In order to isolate and study a single biomembrane transport activity for a substrate, one frequently needs precisely to control

8

1. Importance of Biomembrane Transport

the concentrations of inhibitors of other processes that compete with the activity to transport the substrate. This control is difficult to achieve in intact organs where the inhibitors and substrate may need to migrate relatively long distances to reach the cell membranes. Moreover, the uptake measured in whole organs may represent a composite of several cell types only one of which is the type of interest. For this reason, investigators frequently chose first to isolate cells or even biomembrane vesicles from the cells and then to characterize their transport in a controlled environment in vitro. The possible physiological significance of the transport processes is then usually discussed in the context of what is known about substrate concentrations in extracellular fluids in vivo. Also considered is how the transport is influenced by signaling molecules and other signaling processes such as changes in membrane electrical potential often measured in isolated cells. In light of these attempts to understand transport in its physiological context, surprisingly little attention has been paid so far to the effects on transport of the physical environment of cells in situ. For example, what immediate effects on biomembrane transport are introduced during isolation and purification of a particular cell type or their biomembranes? Do the characteristics of transport change immediately in some or all types of cells when they are isolated? Or do these characteristics remain relatively stable regardless of what may need to be done to the surrounding environment in order to isolate the cells of interest for further investigation? A. H o w Much Does the Cellular Environment in Vivo Influence B i o m e m b r a n e Transport? A partial answer to the preceding questions comes from the study of amino acid transport in early mouse embryos. Cleavage-stage conceptuses and blastocysts lie in close association with the reproductive tract during development. They are, however, quickly and easily separated from the reproductive tract for a period of about 5 days after conception, at which time blastocysts implant in the uterus. When blastocysts are removed from the uterus a day before implantation, their plasma membrane system B ~ transport activity remains constant for several hours in culture (Van Winkle and Campione, 1987; Van Winkle et al. 1990d). In contrast, blastocysts removed from the uterus a few hours prior to implantation experience a dramatic increase in their system B ~ X-AG and/~-transport activities, whereas system b+2 decreases in activity (e.g., Fig. 1.6). The activities of these transport systems change within a few minutes after embryo isolation, and the changes are complete within about half an hour (Van Winkle and Campione, 1987).

FIGURE 1.6 Changes in the transport activities of systems B ~ and b+2 but not system b ~ upon removal of blastocysts from the uterus just prior to implantation (A). In contrast, no change in system B ~ transport activity is observed when blastocysts are removed from the uterus 24 hr before implantation (B). Changes in activity are statistically significant when they are marked with a double asterisk (p < 0.01) (data from Van Winkle et al., 1990d).

Interestingly, the changes also occur on the same time course in blastocysts within the uterus when it is simply massaged gently with a blunt instrument, whereas no such changes in transport system B ~ activity are observed when the uterus is massaged 24 hr prior to blastocyst implantation. Since transport cannot be measured easily in preimplantation embryos within the reproductive tract, it is unclear how the activities of their transport systems may change when they are removed form the uterus relative to their activities in this initial condition. There is, however, little doubt that some of the activities do change as a result of isolation at least in blastocysts nearing implantation. While the possible physiological implications of these changes in blastocysts nearing implantation is of interest primarily to those of us who study early development, the fact that the changes occur at all should evoke broader interest. We currently study transport primarily by isolating the pertinent cells, biomembranes, or even the transport proteins themselves, and the transport proteins may be expressed in other cells or in proteoliposomes. While such studies produce new insights into the functions of transport proteins, the proteins may not function as they normally do in vivo. For this reason, it is anticipated that new investigations will more frequently involve whole tissues, organs and even intact organisms. Numerous examples of such studies are discussed by Taylor and associates in Chapter 10 of this volume. Here we discuss a few examples of the sometimes surprising place of biomembrane transport in the context of multicellular organisms.

Biomembrane Transport in Context

9

B. Transepitheliai Nutrient Transport May Not Be Equivalent Simply to a Composite of All Pertinent Biomembrane Transport Processes for the Nutrient Most of us attribute a central importance to nutrient transporters in the placenta since in few cases is a need for relatively massive transfer of nutrients so conspicuous. While the importance of biomembrane transport to normal placental functioning is difficult to deny, findings with intact animals have shown that the mechanism of transfer of nutrients across the placental trophoblast can be much more complex than anticipated. The simplest way for organic and inorganic solutes to traverse the biomembrane barriers between mother and fetus appears to be for the nutrients to be taken up against their gradients by transporters in the microvillous membrane, for them to then diffuse across the cytosol of the placental trophoblast, and finally for them to migrate out of the cells via transport proteins in the basal membrane. Consequently, much study has focused on identifying and characterizing transport systems and proteins in the two membranes and attempting to envision how the transport processes could be coordinated to catalyze net flux toward the fetus. When this flux is studied in intact animals, however, we quickly learn that we must understand how biomembrane transport fits into a much broader biochemical context, if we are to understand how vectorial nutrient transfer actually Occurs.

For example, only about 38% of the leucine, 11% of the glycine, and none of the serine appearing in the blood plasma of fetal sheep gets there through direct transfer from mother to fetus across the placenta. Rather, amino acids released during placental and fetal protein degradation and nonessential amino acids synthesized in placental and fetal tissues provide most of the amino acids appearing in fetal blood (Geddie et al., 1996). Glycine is synthesized primarily from serine in the placenta for transfer to the fetal circulation (Fig. 1.7), whereas serine is synthesized from glycine and other substrates in the fetal liver (Thureen et aL, 1995). A little over half of the leucine released from the placenta to the fetus appears to arise from placental protein degradation (Ross et aL, 1996). The possibility that the sheep placental trophoblast may also take up and degrade maternal plasma proteins apparently has not been ruled out formally, although the trophoblast in the chorioallantoic placenta appears to have a relatively low endocytic capacity at least in the rodent (Pratten and Lloyd, 1997). In contrast, epithelial cells of the rodent visceral yolk sac placenta display prominent endocytosis and could

FIGURE 1.7 Glycine and serine transport and metabolism in the ovine placenta. While some glycine is transferred directly to the fetus from the mother, most of the glycine appearing in fetal blood plasma from the placenta is produced from serine. Two separate serine pools in the placenta appear to be derived from maternal and fetal sources, and both of these pools are used to produce glycine (adapted from Geddie et aL, 1996 with permission from W. B. Saunders Company Ltd.)

conceivably take up proteins in order to supply amino acids to the embryo/fetus beginning just after implantation and continuing until parturition. 5 Uptake of nutrients by this route clearly permits postimplantation rat embryos to grow at their normally rapid rate even in culture when the epithelium is in direct contact with proteins in the medium (e.g., Beckman et al., 1990, 1991, 1994, 1996, and 1997). It is less clear, however, whether plasma proteins actually reach the yolk sac epithelium in large enough quantities in vivo to contribute significantly to the amino acids reaching the embryo/fetus. If plasma proteins are the principal source of amino acids for the embryo/fetus in vivo, however, then the mechanism of such nutrition in the rodent is significantly more complex than previously anticipated. From about the time of implantation until organogenesis is nearly complete, the epithelium of the yolk sac placenta would take up maternal plasma proteins, degrade them in lysosomes, and then release the resultant amino acids to the embryo. For the amino acids to reach the embryo they would first be transported out of the lysosomes and epithelial cells via amino acid transport systems apparently expressed selectively in the lysosomal and plasma membranes (e.g., Pisoni and Schneider, 1992). Even after the chorioallantoic placenta becomes functional during the latter half of gesta5Two prominent placentas (i.e., the yolk sac and the chorioallantoic placentas) appear to transfer nutrients to the embryo/fetus of several rodent species during a major portion of their gestation, whereas most other eutherian species transfer nutrients primarily or exclusively via the chorioallantoic placenta.

|0

1. Importance of Biomembrane Transport

tion in the rodent, the major source of amino acids to the fetus may still be via the yolk sac placenta (Beckman et al., 1994, 1997). If such is the case, then amino acid transport via the chorioallantoic placenta may not serve primarily for net transport of amino acids to the fetus. Rather, this organ may be viewed better as regulating amino acid levels in the fetus. It may even serve for the net flux of some amino acids from the fetal to the maternal circulation depending on the quantity of amino acids supplied to the fetus via the yolk sac placenta, the maturity of fetal organs, and the nutritional requirements of the fetus. Regardless of what conclusions are finally drawn concerning the role of biomembrane transport in the nutrition of fetuses of various species, two fundamental tenets emerge here. First, biomembrane transport is critical to such nutrition as well as to other processes that are required in order to supply nutrients to the tissues and organs of multicellular plants and animals. Nevertheless, the role of biomembrane transport in these processes may not be as simple or as direct as we first envision. Second, the study of biomembrane transport leads us naturally away from excess reductionism, thus helping to insure that its function will eventually be understood in its physiological context. As we have seen, the ways in which biomembrane transport contributes to the normal functioning of multicellular organisms may not be obvious, although its contribution is obviously real. Biocatalysts usually are needed in order for transport to proceed at rates compatible with life. The discovery and characterization of these transport proteins and systems has thus helped to explain the otherwise unexpectedly rapid rates of migration of some hydrophilic substances across biomembranes.

IV. SUMMARY We have seen that the study of biomembrane transport requires us to consider its biophysics and physical chemistry as well as its biology. Moreover, biomembrane transport is central to the functioning of all multicellular organisms regardless of whether it is considered at the subcellular, tissue, or systemic levels of their organization. Similarly, the study of biomembrane transport is as legitimate a component of investigations into mechanisms of development and differentiation as it is into the functioning of fully formed tissues and organs. Hence, there is scarcely a biological journal or a subsection within such journals from the biophysical to the evolutionary levels of investigation that does not contain articles on the subject of biomembrane transport. Such was, however,

not necessarily the case before biomembrane transport became a fully legitimate field of investigation in these academic disciplines. Partly as a result of the establishment of provisional boundaries to the various academic disciplines of biological sciences in the middle half of this century, some students of biomembrane transport saw the opportunity to cross these artificial subdivisions in highly productive ways. Thanks to the efforts of these pioneers, most modern scientists view their own research as pertinent to a wide range of biological disciplines. Research groups that are focused on certain aspects of biology may of course develop within or between institutions as a consequence of common interests. Most of the time, however, these groups are composed of individuals with broad training, only some of which may have been considered part of the academic discipline historically defined for the department in which they happen to find themselves. The recent dissolution of the Physiology Study Section of the U.S. National Institutes of Health (Ehrenfeld, 1998) is but one consequence of this evolution toward multidisciplinary scientific investigations. By analogy, numerous proteins have evolved over billions of years to catalyze transport of solutes and the solvent across the barriers formed by biomembranes. As the demand for different types of transport increased owing to evolution of more numerous as well as more complex species, the needed transport processes also of course evolved (e.g., see Chapter 8). Similarly, as our investigations have led us to a fuller understanding of biology, the disciplinary boundaries that once helped us to define ourselves now help us more easily to recognize various aspects of biology to which our unfolding work may apply. Viewed as part of our cultural evolution, academic disciplines are destined to become extinct because new paradigms better fitted to the scientific milieu are replacing them. One wonders what scientific approach will evolve to render current interdisciplinary and multidisciplinary approaches obsolete. We continue our exposition of the central position of biomembrane transport in biology in Chapter 2. There we consider the physical and chemical natures of biomembrane barriers, their origins, and their fates. It is impossible fully to understand how various transport proteins form, propagate, or dissipate solute and solvent gradients (Chapters 3 to 7) without understanding the nature of the barriers across which they catalyze transport. Moreover the contributions of these biomembrane transport processes to the physiological functioning of cells and organisms is rooted in the physical and chemical nature of biomembrane barriers and how the barriers may change in various physiological and pathophysio-

Summary logical conditions. Hence, an understanding of the physical and chemical nature of membranes is needed to understand both cellular physiology and the physiology of whole multicellular organisms such as those described in Chapter 10. Similarly, the regulatory mechanisms

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needed to coordinate biomembrane transport in cells and in multicellular organisms (Chapter 9) can only be understood fully if one understands the nature of the barrier for which regulated transport is needed in the first place.

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2 I Biomembrane Composition, Structure, and Turnover

1. INTRODUCTION

process of unknown origin may actually be 20 times more rapid than the more obviously directed processes of endocytosis and exocytosis (Farge, 1995). Endocytosis and exocytosis depend on the cytoskeleton for movement of vesicles to and from the plasma membrane, and the cytoskeletal infrastructure influences the motion of membrane constituents. As for the plasma membrane, however, the function of the cytoskeleton should be viewed neither as passive nor simply structural.

For many years it was unclear how phospholipid bilayers only about 5 nm thick could nevertheless be strong enough to withstand the stresses on the plasma membranes of most cells. True, the sizes of most animal cells are small enough (i.e., --~20/zm in diameter) for adhesive forces between water molecules to maintain a more or less spherical cell shape inside a lipid bilayer surface. For this reason, plasma membranes might, as first approximations, be able to lie relatively passively as unreinforced thin lipid barriers at the surface's of cells. A few moments reflection on the requirements of the membranes of most cells in their natural environments leads, however, immediately away from any such notion of a placid existence for most cell membranes. For example, monocytes surrounded by thin membranes are greatly deformed as they migrate between vascular endothelial cells in response to injury or infection (Fig. 2.1). The endothelial cells, on the other hand, must withstand powerful hemodynamic sheer stresses on their lumenal surfaces that would occur at arterial branch points. In fact, when such stresses are excessive, as in hypertension, they seem to initiate or contribute to development of atherosclerosis (Fig. 2.1). While each cell type may have its own specific requirements for movement, reinforcement, and signaling, all eukarocytic cells benefit from the normally inconspicuous cytoskeletal components that support their membrane structures and functions (Fig. 2.2). Cell membranes also face continuous challenge to their integrity from within. An area of membrane about equal to the area of the entire cell surface turns over about every 30 min in many cells due to endocytosis and exocytosis. A less conspicuous vesiculation and refusion

!I. IS THE FLUID MOSAIC MODEL OF MEMBRANE STRUCTURE STILL ADEQUATE? A. The Lipid Bilayer Hypothesis The lipid bilayer is now well established as the fundamental structure of most biomembranes (Fig. 2.3A). Nevertheless, nonlamellar lipid structures also form important components of biomembranes. These structures and their influences on membrane function have been reviewed recently in a volume edited by Richard Epand (1998). For this reason and to conserve space, we focus principally on the lipid bilayer structure of membranes in this chapter. Many phospholipids will assume the bilayer structure spontaneously in membranelike structures known as liposomes under the right experimental conditions either alone or in combination with other lipids (Gregoriadis, 1993). More realistic "snapshots" of fully hydrated artificial phospholipid bilayers (e.g., Fig. 2.3B) have been produced recently through computer simulations (e.g., Jakobsson, 1997). The phospholipids that are present in biomembranes are highly amphipathic; they have phosphate-containing groups esterified to molecules (usually glycerol) that also have long-chain fatty acyl

13

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2. Biomembrane Composition, Structure and Turnover

FIGURE 2.1 Involvement of monocytes and endothelial cells in development of the fatty streak and, eventually, atherosclerosis. Note in particular the physical stresses that monocytes need to place on their own plasma membranes in order for them to pass between endothelial cells and the hemodynamic sheer stresses to which endothelial cells are exposed at arterial branch points. Such sheer stresses would, of course, increase as blood pressure increases.

groups (Fig. 2.3C). The phosphate-containing groups are electrically charged and, hence, highly hydrophilic, whereas the hydrocarbon side chains of the fatty acyl groups are quite hydrophobic. In the phospholipid bilayer, these hydrophobic side chains extend within each leaflet of the bilayer toward each other to form the hydrophobic interior of biomembranes (Fig. 2.3A). In contrast, the hydrophilic phosphate-containing groups seek positions in the bilayer between the hydrophobic interior and either of two external aqueous phases. One surface of the bilayer faces the cytosol of cells, whereas the other surface of the bilayer borders the lumen of

organelles, the interior of membrane vesicles, or the exterior of cells (Fig. 2.3D). The formation of bilayers is primarily an entropydriven process because water exists in an ice-like rather than a liquid state when the water is associated with the hydrocarbon side chains of the fatty acyl groups of the phospholipids. When the hydrocarbon side chains associate with each other in the interior of the lipid bilayer instead of with water, the water can become liquid rather than remain ice-like. It is this greater freedom of movement of water in the liquid state that drives formation of these so-called hydrophobic bonds or, more properly,

Fluid Mosaic Model of Membrane Structure

| 5

FIGURE 2.2 Cell in culture fixed and stained to expose proteins that form the filamentous cytoskeleton (adapted from Alberts et al., 1994, with permission from Garland Publishing, Inc.).

hydrophobic interactions. In the present case, these hydrophobic interactions refer to the sequestration of the hydrocarbon side chains in the interior of the lipid bilayer of biomembranes away from most of the water. Animal biomembranes also contain other lipids, such as cholesterol, in addition to phospholipids. Cholesterol is less amphipathic than are phospholipids because the hydrophilic portion of cholesterol is due primarily to its uncharged and relatively small hydroxyl group rather than to an electrically charged and much larger phosphate-containing group (Fig. 2.3C). For this reason, cholesterol appears in many instances not to be as confined as phospholipids are to one leaflet or the other of the membrane bilayer. It is this greater ability of cholesterol to flip from one leaflet to the other that is

believed by some investigators to permit cells to undergo rapid shape changes without causing one leaflet of their plasma membrane to "wrinkle" and the other leaflet to "gap" (Fig. 2.3E). Cholesterol also migrates from the outer to the inner leaflet of the platelet plasma membrane when these cells are activated apparently owing to migration of phosphatidylethanolamine in the reverse direction (Boesze-Battaglia and Schimmel, 1997). Although clearly valid, the lipid bilayer hypothesis remains an active field of investigation. For example, we are still attempting fully to understand the consequences of the asymmetric distribution of lipids across the bilayer (see below). In addition, we are only beginning to appreciate how the existence of most membrane

16

z. Biomembrane Composition, Structure and Turnover

lipid bilayers in a liquid-crystalline state (defined here as the state of transition between the wholly liquid and wholly crystalline phases) contributes to their function. 1 The complex composition and asymmetric distribution of lipids in the leaflets of the membrane bilayer probably contributes to the relatively wide range of temperatures over which the membrane "melts" (Fig. 2.4). The continuous transition of lipid in membranes between the liquid and crystalline states creates in membranes transient domains that undoubtedly influence not only the structure but also the function of membrane constituents. Because membrane constituents may be more concentrated in one type of domain than in the other, the influence of these transient domains on membrane function may be quite different from the effects on function of wholly liquid or wholly crystalline bilayers. For example, what is the consequence to, say, a glutamate transport protein molecule when it is present in a crystalline vs a liquid domain of different lipid compositions? Could existence in one or the other domain influence whether the protein functions in some instances as a glutamate transporter and in other instances as a C1- channel? (See Sections III and IV of Chapter 6 for further discussion of such multiple transport functions of these proteins.) In addition, many integral membrane proteins appear to be associated preferentially with either liquid or crystalline domains depending on the domains for which the protein molecule has greater affinity (Marsh, 1995). The sizes of crystalline domains are larger than are liquid ones, at least in artificial bilayers, apparently owing to a smaller number of nucleation sites in the former case (Sankaram et al., 1992). Such differences in the sizes of the liquid and crystalline domains as well as the preferential association of different proteins with one or the other of the domains likely influences the interactions among protein molecules in the bilayer. 1The terminology in the literature is somewhat unclear in regard to what is meant by the liquid-crystalline state of the lipid bilayer, perhaps because the existence of phase separation in the bilayer has only recently gained wider acceptance (e.g., Brown and London, 1997). Here we define the wholly solid, gel, or crystalline state as the state of the bilayer before it begins to melt, whereas the wholly liquid or fluid state is defined as the state of the bilayer after it has melted. At physiologically normal temperatures the membrane lipid bilayer exists between these two states in what is termed here the liquid-crystalline state. Phospholipids in the liquid-crystalline state of membranes may be viewed as highly ordered, as in a crystal, and yet highly mobile, as in a liquid. The same is to some extent also the case, however, for the lipid in bilayers just above or just below their melting temperatures. The lipid is simply more ordered in the crystalline state and more mobile in the liquid state. Hence, what is perhaps more important to appreciate about the lipid in biomembranes is that the order and

mobility of the lipid varies with location in the membrane; some such transient domains appear to be wholly crystalline while others appear to be fully liquid. The possible importance of melting and freezing of these transient domains to the physiologicalfunctioning of biomembranes is discussed in this and subsequent chapters.

FIGURE 2.3 Lipid bilayer structure ofbiomembranes. (A) The lipid bilayer in which circles represent charged phosphoryl-containing portions of the lipid molecules and hydrophobic hydrocarbon chains extend toward the center of the bilayer. (B) Computer simulation of a fully hydrated artificial lipid bilayer in which the phosphoryl-containing portions of the lipid molecules can be seen to mix on a molecular level with water molecules. Hydrogen atoms in water molecules are shown in white, and the oxygen atoms in water are somewhat lighter in color than atoms in phospholipid head group. The sizes of the water molecules and phospholipid head group atoms have been reduced in order to see into the structure (adapted from Chiu et aL, 1995, with permission from The Biophysical Society). (C) Details of the structures of several membrane lipid molecules as they would be aligned in one layer of the bilayer. The zigzag lines represent hydrocarbon chains of various lengths (adapted from Finean and Miche 11,1981, with permission from Else vier Science). (D) One surface of the bilayer always faces the cytosol, whereas the other surface may face the lumen of intracellular organelles

(except mitochondria and peroxisomes), the inside of membranebound vesiclesorthe extracellular environment (adapted from van Helvoort and van Meer, 1995,with permission from Elsevier Science). (E) Cholesterol molecules in the bilayer may flip from one layer to the other when a shape change is needed in the membrane, thus helping to prevent formation of gaps and wrinkles in either layer.

It might, at first, seem to be a simple matter to study the effects of crystalline and liquid domains in membranes on their function by studying the membranes below or above their melting temperatures. It can be shown, however, that the fluid (i.e., entirely liquid) and gel (i.e., entirely crystalline) states of membrane lipids are probably not equivalent to liquid and crystalline lipid domains in membranes in phase transition. For example, triiodothyronine decreases the freedom of lipid motion in artificial membranes in the fluid state but increases it in the liquid-crystalline and gel states (Farias et aL, 1995). A possible explanation for this phenomenon is that the hormone is partitioned mainly to the crystalline phase of the liquid-crystalline state, whereas no such partitioning is possible in the fluid state. If, however, partitioning occurs in the liquid-crystalline state but it cannot by definition occur in the fluid or gel states, then this phenomenon becomes itself proof that the transient crystalline and liquid lipid domains in membranes in phase transition are unlikely to have a composition and structure that are identical to those of membranes

Fluid Mosaic Model of Membrane Structure

FIGURE 2.3

wholly in the gel or fluid state. If triiodothyronine is partitioned to the crystalline domains then it is not evenly distributed in bilayers in the liquid-crystalline state, whereas it would be evenly distributed in the wholly fluid or wholly gel states. Preferential association of some lipid species with particular integral membrane proteins may also result in partitioning of the lipid into a liquid or crystalline domain since the lipid species attracted to a protein molecule may itself be more likely to be in one or the other of these states (Marsh, 1995). The lipid compositions of liquid and crystalline domains in mem-

|7

(Continued)

branes can be studied using X-ray microanalysis (Hui, 1995), and electron microscopy helps to determine the geometries of the domains (e.g., Fig. 2.5). As just discussed, however, it becomes a difficult problem indeed to study the effects of different domains on the functions of proteins that intrude into the lipid bilayer. In fact, it is even conceivable that some proteins require the crystalline or liquid lipid domain in which they exist at a given moment to "melt" or "freeze," respectively, in order for the proteins to complete their functions (e.g., see Section VI of Chapter 3 regarding the functioning of Na+K+ATPase).

|8

2. Biomembrane Composition, Structure and Turnover

/

C

> > > > >

OH

c=o 9.o O O CH2-CH 2 CH2

C.Oo C.Oo

~.o ~.o O 9 CH=-CH 2 CH2

(~"O ~ C(=O (~ 9 (~=O 9 'C=O ,O !~O ~C= CH2- CH2 CI~ CH2-cH 2 CH2- CH2 Cholesterol CH2 HO ,CH2 ~H2 H C~ ~H2 C,H= ,CH= ,CH~ 0 9. O ,O 9 9 O=P-O O=P-O" O~P-O" o.,P-O o'-f=oo=,P-O" O-P-O'o H O O H ~ ~ HOOH2-~~OH~ ,o o 0 ~H2 H C]~2 /CH2 CH~ c.o ,c CH~ H~OO'O ~'O'O'O'O'O'O H 'O' 0 CH C,H2 CH O O CH3 (i; nln3 NH3* CH~",I~-CH3 CH~'N(~i)~CH3 6H HOCH " .0 CH3 CH3 Diphosphaditylglycerol ( DPG ) P PhosphatidylSphingomyelin Galactosyl O" ",r-NHC~, ( SM ) ceramide HOCH~o ~ CH3 inositol( PI ) serine( PS ) ethanolamine choline ( PC ) ( cerebroside) 0 (PE) !

!

!

.o o. I

!

o !

~--~'%L,=,

+

HOCH~o ~OOH Monosialoganglioside ( GMj )

Fluid Mosaic Model of Membrane Structure

FIGURE 2.4

19

Phase transition of lipids in the membranes of living

Acholeplasma laidlawii cells (circles) and in the membranes isolated

FIGURE 2.3 (Continued)

from these cells (triangles). According to the terminology used here, the bilayers are in the gel state at temperatures along the lower plateaus and they are in the fluid state at temperatures along the upper ones. At temperatures between the plateaus the bilayers are in the liquid-crystalline state. The broad range of temperatures over which the membrane "melts" and, hence, at which both liquid and crystalline lipid domains are present appears to be due in large part to the presence of integral membrane proteins in the bilayer and that these proteins associate preferentially with some lipid species (e.g., Marsh, 1995). Asymmetric distribution of lipids across the bilayer and heterogeneity of fatty acid residues that are saturated, monounsaturated, or polyunsaturated also probably contributes to the characteristics of the membrane lipid phase transition (adapted from Mantsch and McElhaney, 1991, with permission from Elsevier Science).

B. Protein Intrusion into the Bilayer The protein content of biomembranes varies from about 20% to more than 70% depending on the membrane. Proteins can also be associated with membranes peripherally as well as being integral components of them (Fig. 2.6). Biomembrane transport is generally believed to be catalyzed by integral membrane proteins, although their activities may be profoundly influenced by peripheral proteins. Integral proteins have one or more membrane-spanning segments. Most such segments are believed to be c~-helices of about 21 consecutive mainly hydrophobic residues oriented more or less perpendicularly to the plane of the membrane (von Heijne, 1994). Some of the helices may, however, be tilted, and they may even be parallel to the membrane in some cases (Persson and Argos, 1996). In addition, B-barrel components of some integral membrane proteins have been proposed to traverse the plasma membranes of eukaryotic cells (Fischbarg and Vera, 1995) as well as the outer membranes of Gram-negative bacteria and mitochondria (see summary of outer membrane porins in Chapter 8 of this volume). While the trans-

membrane component of the GLUT1 glucose transport protein is shown to be all /3-barrel in Fig. 2.7, more recent data are consistent with the possibility that it is composed of a mixed structure (Ducarme et al., 1996). In this mixed model, 10 c~-helices and 4/3-strands of GLUT1 are proposed to traverse the plasma membrane. The tertiary and even quaternary structures of several integral membrane proteins have also been studied in some detail. For example, the aquaporin-1 (CHIP28) monomer, a member of the protein family that forms water channels in numerous epithelial and nonepithelial tissues (Verkman et al., 1996), has been observed using cryoelectron crystallography to form tetrameres of four water channel pathways through the plasma membrane (Fig. 2.8). Each monomer forms a water channel apparently surrounded by six transmembrane c~-helices. In the case of acetylcholine-gated inorganic ion channels, the channels have even been imaged in different closed and open conformations (Unwin, 1995). In spite of these impressive advances, the structures of most integral membrane proteins are still only slowly being described in detail. Moreover, while our ability both to predict

20

z. Biomembrane Composition, Structure and Turnover

FIGURE 2.5 Freeze-fracture electron micrograph of one face of a phospholipid bilayer. Crystalline and liquid domains appear to be associated with characteristic surface undulations that help to distinguish them (Bar = 500 nm) (adapted from Hui, 1995, with permission from Taylor & Francis, London, UK).

topologies of membrane transport proteins and to test these predictions is improving (e.g., Jones et al., 1996; Persson and Argos, 1996), the detailed biochemical and biophysical mechanisms by which the proteins catalyze biomembrane transport, and the mechanisms of bioenergetic coupling of transport processes to each other or

to chemical change, remain, in most instances, very active areas of research. In spite of our still incomplete understanding of these transport mechanisms, many investigators assume some fundamental knowledge of the laws that govern them. For example, although transport proteins have asymmetric orientations in biomembranes, most investigators believe that the proteins catalyze thermodynamically symmetric transport in instances where the transport is not coupled to a conspicuous source of free energy. However, such may not always be the case, as discussed in several of the following chapters (e.g., Section II,C,2 of Chapter 7). C. B i o m e m b r a n e Structure Is A s y m m e t r i c as Well as H e t e r o g e n o u s 1. Proteins

FIGURE 2.6 Fluid mosaic model of biomembrane structure. Both integral and peripheral protein molecules are associated with the lipid bilayer, and the protein as well as the lipid molecules themselves diffuse laterally in the bilayer. In addition, the lipids near proteins are shown to be slightly disturbed relative to the more regular order in the rest of the bilayer. Actually, the lipid should be composed of more ordered "crystalline" domains and less ordered "liquid" domains, and some proteins should be shown as increasing order while

others decrease it (see text).

The mechanism of insertion of protein molecules into eukaroytic biomembranes appears to insure that each copy of a particular protein will have the same asymmetric orientation in a membrane (see also Section IV,B below). Most integral membrane proteins are inserted into membranes in the endoplasmic reticulum by a process that involves amino acid residue signaling sequences (Fig. 2.9). Although much is known about the topological information in the protein to be inserted, we are still learning how the cell decodes this informa-

Fluid Mosaic Model of Membrane Structure

2 |

2. Lipids

FIGURE 2.7 Possible structure of the GLUT1 glucose transport protein molecule. Note the considerable B-barrel structure (arrow) that has been proposed to span the plasma membrane. This protein appears to catalyze the transport of water as well as of glucose (adapted from Fischbarg and Vera, 1995, with permission from the American Physiological Society).

tion (von Heijne, 1994). Moreover, the number of transmembrane segments may change after initial insertion of proteins into the membrane as is the case for aquaporin-1 (CHIP28) monomers during trafficking from the endoplasmic reticulum to the plasma membrane (Verkman et aL, 1996). Hence some membrane transport proteins may undergo relatively large conformational changes during processing and even while functioning. (See detailed discussion of specific examples of transport protein function in Chapters 5 to 7.) Nevertheless, most transport and other integral membrane proteins likely retain their asymmetric orientations after insertion into biomembranes.

While integral membrane proteins have asymmetric orientations, lipids have asymmetric concentrations across biomembranes. For example, among the four most abundant categories of phospholipids in plasma membranes, the anionic one (phosphatidylserine) and a zwitterionic one (phosphatidylethanolamine) are usually more concentrated in the inner than in the outer leaflet of the bilayer, whereas the converse is true for the other zwitterionic phospholipids (sphingomyelin and phosphatidylcholine) (Table 2.1). More rapid movement of phosphatidylserine and phosphatidylethanolamine from the outer to the inner leaflet than the reverse is catalyzed by a membrane-bound MgZ+ATPase (Zachowski, 1993; Auland et al., 1994). Free energy is required to move these phospholipids against their concentration gradients and, in the case of the anionic phosphatidylserine, against the inside negative membrane electrical potential. Similarly, the outward movement of phosphatidylcholine is two to three times more rapid than inward migration (Zachowski, 1993), and stimulation of this difference by cytosolic ATP indicates that the outward transport may also be catalyzed by an ATPase. In contrast, the location of sphingomyelin in the outer leaflet appears to result from its synthesis in the lumen of the cis-Golgi through transfer of a phosphocholine residue from phosphatidylcholine (leaving diacylglycerol) to ceramide (see also Section IV,C below). Since little or no movement of sphingomyelin from the outer to the inner leaflet has been observed in healthy cells, this phospholipid appears to remain in the outer leaflet after it is synthesized. Although more sphingomyelin may be synthesized from ceramide and phosphatidylcholine in recycling endosomes than in the cis-Golgi in some cells (Fig. 2.10), its exclusive synthesis in the outer leaflet still appears to account for its asymmetric distribution in the plasma membrane. The asymmetric distribution of lipids across the plasma membrane is scrambled by several normal as well as artificial processes. Cellular activation by a variety of stimuli is associated with an increase in the cytosolic free C a 2+ concentration. Since C a 2+ in the cytosol inhibits MgZ+ATPase, it has been proposed that inhibition of this enzyme also leads to the increase in the concentrations of phosphatidylserine and phosphatidylethanolamine in the outer leaflet of platelet cell membranes during activation. Inhibition of MgZ+ATPase by N-ethylmaleimide does not, however, result in redistribution of phosphatidylserine to the outer leaflet of the platelet plasma membrane (Bas~e et aL, 1993). Hence, scrambling of lipid asymmetry during cell activation may involve more than simply inhibition of

22

2. Biomembrane Composition, Structure and Turnover

FIGURE 2.8 Projection structure of the aquaporin-1 channel-forming integral membrane protein molecule of 28 kDa (CHIP28) in the membrane at 6 A resolution by cryoelectron crystallography. The protein appears to be a tetramer of four 28-kDa monomers, each of which forms a water channel. The putative, channelforming transmembrane a-helices are numbered 1 to 6 in one monomer (Bar = 10A) (adapted from Mitra et al., 1995, with permission from Nature Structural Biology).

Mg2+ATPase by Ca 2+, at least in platelets. In this regard, membrane fusion events such as endocytosis and exocytosis also probably lead to local transient scrambling of lipid asymmetries (Zachowski, 1993). The initial movement of phosphatidylserine from the inner to the outer leaflet appears to precede vesicle shedding by platelets (Basge et al., 1993). Hence, it is more likely that cortical granule exocytosis rather than subsequent

vesicular budding contributes to the scrambling of phospholipid asymmetry during platelet activation. This mechanism also would account for the movement of sphingomyelin from the outer to the inner leaflet, a movement which does not appear to occur by other biochemical means in most biomembranes. In addition, a Ca2+-dependent "scramblase" appears to catalyze the degradation of the phosphatidylserine and phosphati-

Fluid Mosaic Model of Membrane Structure

23

FIGURE 2.9 Synthesis and insertion of protein molecules into the lumen or membrane of the endoplasmic reticulum (ER). (A) Although the simplified diagram is for insertion of a protein into the lumen of the ER, the presence of multiple, uncleaved start- or stop-transfer signal peptides in the primary structure of a protein presumably can lead to insertion of a multipass, integral membrane protein molecule as in B. (B) Hypothetical model for insertion of a double-pass protein molecule in the ER membrane (adapted from Alberts et al., 1994, with permission from Garland Publishing, Inc.).

dylcholine concentration gradients across the platelet plasma membrane (Comfurius et al., 1996) as well as the plasma membranes of other human cells (e.g., Zhou et al., 1997). Regardless of the mechanism, the movement of phosphatidylserine to the outer leaflet has important physiological and pathophysiological consequences (reviewed more extensively by Zachowski, 1993). In platelets, phosphatidylserine in the outer leaflet favors conversion of coagulation factor X to Xa and the association of factor Xa with factor Va. These changes help to generate a catalytic surface that promotes coagulation. Similarly, abnormal red cells, such as ones that are sickled, adhere more strongly to vascular endothelial cells perhaps as a consequence of the greater concentrations of phosphatidylserine in their outer leaflets relative to normal cells. Also as a consequence of greater external phosphatidylserine exposure, apoptotic lymphocytes and some tumorigenic cells may be destroyed more readily by monocytes or macrophages. Differences in phospholipid composition of the inner and outer leaflets of erythrocytes and quiescent platelets has been used successfully to design hemocompatible surfaces (Chapman, 1993). Phosphatidylcholine coating reduces the adsorption of fibrinogen and platelets to artificial surfaces and prevents platelet activation by the surfaces. These surfaces should be useful in the production of better artificial blood-contacting devices including catheters, indwelling biosensors, extracorporeal circuits, and filtration membranes. It has also been proposed that the process of forming asymmetric distributions of phospholipids in the plasma membrane has itself a function independent of the func-

tions of the asymmetrically distributed phospholipids themselves or subsequent scrambling of their asymmetric distribution (reviewed by Williamson and Schlegel, 1994). In this view, the transport of the phospholipids from the outer to the inner leaflet would create a force in the inner leaflet which bends the membrane inward. Such is almost certainly the case in erythrocytes where excessive transport of phosphatidylserine and phosphatidylethanolamine from the outer to the inner leaflets results in the formation of stomatocytes (i.e., mouthlike cells) (reviewed by Zachowski, 1993). In cells with a less restrictive cytoskeleton, the transport of phospholipids from the outer to the inner leaflet could conceivably contribute to invaginations of the membrane such as those that occur during endocytosis. Whether phospholipid transport p e r se contributes significantly to formation of endocytic vesicles or to other membrane processes remains to be established experimentally.

3. Carbohydrates Unlike proteins and lipids, carbohydrates are not considered to be an integral component of membranes. Their peripheral association with biomembranes is, however, highly asymmetric. Oligosaccharides are covalently bound to membrane lipids and integral proteins only on their noncytosolic sides. Oligosaccharides are assembled, transferred to membrane proteins or lipids, and subsequently modified in the lumen of the endoplasmic reticulum and Golgi apparatus (Fig. 2.11). No mechanism is known for the assembly and attachment of these molecules to segments of the proteins or lipids at the cytosolic surfaces of membranes. The resultant

24

2. Biomembrane Composition, Structure and Turnover

TABLE 2.1 Percentage of Each Main Phospholipid Class Present in the Outer Leaflet of Various Animal Plasma Membranes a Cell

Human erythrocyte Mouse erythrocyte Rat erythrocyte Monkey erythrocyte Human platelet Pig platelet Mouse erythroleukaemic cell LM cell Mouse synaptosome Rabbit intestinal brush border Rabbit kidney brush border Trout intestinal brush border Middle Posterior Rat cardiac sarcolemna Krebs ascites cell Rat hepatocyte Bile canalicular surface Contiguous surface Sinusoidal surface Chick embyro fibroblast Chick embryo myoblast Quail embyro myoblast

Sphingomyelin

PC

PE

PS

80 85 100 100 82 93 91 80

77 50 62 63 67 45 40 45 48

-- 2/3.

(3.26)

Note, however, that the more these ratios exceed 2/3, the more the membrane electrical potential calculated from Eq. (3.25) decreases in magnitude (compartment B is negative). In contrast, the membrane electrical potential as assessed by the actual numbers of charged ions present in each compartment increases in magnitude (compartment B is still negative) as the ratios exceed 2/3. For example, if, say, 24 instead of 27 Na § ions were present in compartment B, then, of course, 21 Na § would be in A, 16 C1- would be in A and 14 C1- would be in B. In this case, the total negative charge in B would be - 2 9 while the positive charge in this compartment would be + 24. Hence, the magnitude of the membrane electrical potential based on assessment of the

Gibbs-Donnan Effect Generates Osmotic Pressure

47

m e m b r a n e potential that is p r o d u c e d in living cells by only a slight gradient in total charge (roughly 1 part in 1000; Hille, 1992) nevertheless contributes a b o u t as much to the total chemical potential difference of N a § ions or K § ions across the m e m b r a n e as do the m o r e than 10-fold differences in their activities and concentrations.

V. T h e G I B B S - D O N N A N EFFECT A L S O GENERATES O S M O T I C PRESSURE

FIGURE 3.5 The system in Fig. 3.4 after it reaches the equilibrium defined by Eq. (3.25). Although the total numbers of negative and positive charges are shown to be equal on both sides of the membrane, compartment A is actually slightly more positive than negative, whereas the reverse is true for compartment B. For example, it can be assumed that the compartments each have a volume of 1 ml, the membrane has an area of 1 cm2 and the 18 Na + in A, 27 Na + in B, 12 C1- in B, and 18 CI- in A actually represent activities of 0.18, 0.27, 0.12, and 0.18 M, respectively. In this case, the ratios of 0.18 M/0.27 M for Na + and 0.12 M/0.18 M for C1- between the two compartments need to be offset by about one billionth in order to produce the transmembrane electrical potential of -10 mV that must be present according to Eq. (3.25). The pressure on the solution in compartment B also must be higher than that on the solution in A to produce equilibrium (see Section V of the text).

actual charged ions increases from zero as the ratio exceeds 2/3, w h e r e a s the m a g n i t u d e of this electrical potential should decrease according to Eq. (3.25). T h e solution to this a p p a r e n t p a r a d o x is that the ratios of the N a + and C1- activities in Eq. (3.26) exceed 2/3, but only by a very small amount. Ratios of 2/3 can be used in Eq. (3.25) to calculate an electrical m e m b r a n e potential of - 1 0 m V at 298~ ( S = 23,061 cal V -1 equiv -1 and R = 1.987 cal deg -1 mol-1). It can be calculated for the m e m b r a n e depicted in Fig. 3.5 (assuming that 15 N a + or C1- = 0.15 M activity of these ions) that the 2/3 ratio would n e e d to be offset by only about one billionth ( 1 0 -9 ) t o p r o d u c e an electrical m e m b r a n e potential of 10 m V if the m e m b r a n e has an area of 1 cm 2 and the v o l u m e of each c o m p a r t m e n t is 1 ml (see similar calculation for living cells in Hille, 1984). Conversely, the r e a d e r may also now begin to appreciate that very small total ion activity gradients p r o d u c e electrical m e m b r a n e potentials that have relatively large effects on the total chemical potentials of ions. For example, we show in Section V I , A below that the electrical

In addition to the generation of an electrical potential difference across m e m b r a n e s , a n o t h e r c o n s e q u e n c e of the G i b b s - D o n n a n effect is to p r o d u c e osmotic pressure. Osmosis is defined here as the migration of water from a place of higher total chemical potential of water to a place of lower total chemical potential of water through a m e m b r a n e p e r m e a b l e to water but not all solutes. 3 As for solutes, the free energy of water in a c o m p a r t m e n t can be expressed as its chemical potential, /Xs, w h e r e /Xs =/X~ + R T l n as.

(3.27)

Since water molecules are u n c h a r g e d (z = 0), the m e m b r a n e potential does not directly influence its total chemical potential. Nevertheless, by influencing the total chemical potential of anions and cations in the system, the m e m b r a n e potential can have a significant indirect effect on the total chemical potential of the solvent. F o r the example illustrated i n Figs. 3.4 and 3.5, it can be seen that at equilibrium of the solutes, the total n u m b e r (or activity) of dissolved solute particles in c o m p a r t m e n t B is 27 Na + + 12 C1- + 1 A 15- = 40, whereas the total in compartm e n t A is 18 Na + + 18 C1- = 36 (Fig. 3.5). This difference is due both to the i m p e r m e a n t anion and to its consequences for the distributions of Na + and C1- across the m e m b r a n e . T h e difference in the total activities of solutes in c o m p a r t m e n t s A and B also m e a n s that the system r e p r e s e n t e d in Fig. 3.5 is not yet at equilibrium in regard to the solvent. Let's assume that no force is at work to maintain equal volumes in c o m p a r t m e n t s A and B. In this case, we could a t t e m p t to allow the system to reach equilibrium by allowing water to m o v e from c o m p a r t m e n t A to c o m p a r t m e n t B, thus making the volume of B g r e a t e r than A. T h e r e is, however, an i m p e r m e a n t ion in com-

3The reader may notice that we are careful not to state or imply that water or relatively small hydrophilic solutes actually migrate through most biomembranes by ordinary diffusion. Nevertheless, the thermodynamic expression for the overall process is not altered if it is assumed that water moves between compartments A and B by that simple process. Similarly, our convenient assumption here that a membrane may be permeable to water or solutes should not be taken to mean that permeation occurs by diffusion.

48

3. Thermodynamics and Transport

partment B, and its presence there also results in unequal distributions of permeant ions between compartments A and B (see above). If water moved from compartment A to compartment B, it would tend to concentrate the solutes in A and dilute them in B (Fig. 3.6). To reestablish the appropriate ratios of the activities of Na + and C1- according to Eq. (3.25), each of these ions would also move from A to B. Hence, if water were allowed to move freely from A to B until it reached equilibrium, it would continue to do so until all of the water and permeant ions had moved from A to B. To maintain constant volumes of compartments A and B, it is necessary to establish an equilibrium by allowing pressure, rather than volume, to change. Pressure (P) and volume (V) can be introduced into the expression for the total chemical potential of water, txts, by expanding Eq. (3.27) as follows

constant pressure that has been assumed until now to be adequate for our considerations of total chemical potential. For the system depicted in Fig. 3.5, it is possible to stop the net migration of water molecules from A to B and establish equilibrium by applying enough pressure to the solution in compartment B to stop the net migration of water. At equilibrium

where /.LtSA and lxtsB are the total chemical potentials of water in compartments A and B, respectively. Similarly, we can write from Eq. (3.28) and (3.29) /X~ + R T l n aSA + V s ( P A - P o ) = ~~ + R T l n aSB + Vs (PB - Po), which simplifies to I

PA-

PB = ( R T / V s ) ( l n

I

~ts = ~t/'S -+" Vs (P - Po)

and I

/.Lts = /.L~ -t- R T l n as + Vs (P - Po),

(3.28)

where Vs is the volume occupied by one mole of water, P is the pressure applied to the solution, and Po is the

(3.29)

/.LtSA = ~tSB ,

(asB/aSA)),

(3.30)

where the subscripts A and B indicate that the parameters are for the solutions in compartments A and B, respectively. The difference between PA and PB is the amount by which the pressure on compartment B must exceed that on A to stop the net migration of water. More generally, equations such as Eq. (3.30) can be developed for any water solution in comparison to pure water to define the osmotic pressure, 7r, of the solution. The osmotic pressure of a solution is defined here as the amount of pressure that must be applied to the solution in order to stop the net movement of water into it from a compartment of pure water through a semipermeable membrane. For the solutions in compartments A and B of Fig. 3.5, respectively ~'A : ( R T / V s ) ( l n

(aw/asA)),

and 7rB = (RT/Vs)(ln (aw/asB)), where aw is the activity of pure water. Hence, for the system in Fig. 3.5 involving compartments A and B 7rA -- 7rB = ( R T / V s ) ( l n

FIGURE 3.6 Effect of the equilibrium depicted in Fig. 3.5 on the net migration of water (represented by the solid arrows) between the compartments. Because the total activities of solutes in compartment

B are greater than in compartment A, the activity (and total chemical potential) of water is greater in A than in B. Hence, water and, as a result of the water movement, Na+ and C1- would all tend to move from A to B. The movement of water and ions from A to B would continue until compartment A disappears, unless enough pressure is applied to the solution in B (in excess to that applied to the solution in A) to stop the net migration of water.

(asB/aSA)).

(3.31)

The difference between the osmotic pressures of the two solutions, ~'A -- 7rB, is, o f course, the degree to which the pressure that is applied to the solution in compartment B must exceed the pressure on the solution in compartment A in order to stop the net migration of water molecules from A to B, which is the same as the difference between P A and PB in Eq. (3.30). Another way to stop the net m o v e m e n t of water from A to B without applying different pressures to the two solutions is to establish a steady state rather than an equilibrium. In this case, pressure and volume can again remain constant while the excess of permeant ions in

49

Chemical Reactions Drive Primary Active Transport

compartment B is reduced by their active transport to compartment A. Active transport of C1- from B to A would not only tend to equalize the osmotic pressures of the two solutions, but it would also reduce or reverse the membrane electrical potential. In contrast, the active transport of Na + from B to A would both counteract the difference in osmotic pressure and increase the existing membrane electrical potential. Active extrusion of Na + from compartment B is analogous to the action of Na + K+ATPase and other such primary active transport processes in the plasma membranes of most cells. In the case of Na+K+ATPase, the extrusion of more monovalent cations from the cell than are taken into the cell from the extracellular fluid increases the inside negative electrical potential across the membrane. In addition, this transport by Na+K+ATPase helps to reduce the cellular swelling that would otherwise occur as a result of the GibbsDonnan and other effects (see also Section X below).

VI. CHEMICAL REACTIONS DRIVE PRIMARY ACTIVE TRANSPORT

A. C o m p a r i s o n s of the Free Energies of Cation Transport and ATP Hydrolysis That Are Catalyzed by Na+K+ATPase In contrast to the hypothetical example just described, the concentration and activity gradients of most solutes across the biomembranes of living cells usually lie in the same direction as their total chemical potential gradients across these membranes. Hence, in most real examples of primary active transport, solutes are moved against their concentration and activity gradients as well as against their total chemical potential gradients. In the case of Na+K+ATPase, the endergonic processes of Na § extrusion from cells and K + uptake by them are driven by the exergonic process of ATP hydrolysis to ADP and Pi. Separate thermodynamic expressions can be derived for each of these coupled processes. To derive an expression for the transport of Na +, the intracellular and extracellular compartments may be distinguished with "i" and "o," respectively. Equation (3.17) previously derived for a compartment that was designated A /d,tNa+A ---- /.s176 + nt- R T In aNa+A + ZNa+ ~ " ~I'tA

We have seen that the total chemical potential of a solute may depend on its charge, the electrical membrane potential, and other permeant and nonpermeant ions in the system. In the hypothetical example just described, the active transport of two Na + ions from compartment B to compartment A in Fig. 3.5 would reestablish osmotic balance. This endergonic process actually decreases the Na § ion activity gradient in this case. The total chemical potential of Na + is, however, also dependent on the membrane potential against which Na + must be moved in going from B to A. Hence, the active movement of Na + from B to A in Fig. 3.5 represents formation of a gradient of the total chemical potential of Na § such that

From Eq. (3.17) it can be seen that the movement of two Na + ions from B to A would increase /./,tNa+A by increasing both the R T l n aNa+A and the Zya+ S ~A terms -'1- R T In aNa+A + ZNa+ f

'(I)'A,

(3.17)

where aNa+A would increase as would the magnitude of ~A. Similarly, from Eq. (3.19) it is clear that p, tNa+B would decrease due to a decrease in both aNa+B and ~B (i.e., ~B would become more negative) /ZtNa+B "-- /d,~

may then be rewritten /.LtNa+i- /.s176 + nt- R T l n aNa+i + ZNa+f ~I/'i

(3.32)

for the total chemical potential of Na § inside cells and /d,t N a + o -

/d,~

q- R T In aNa+o -}- ZNa+ ~"XI~o

(3.33)

for the total chemical potential of Na § outside them. The total chemical potential gradient against which Na § must be moved and, hence, the free energy change for extrusion of Na § from cells can now be written as the difference between the total chemical potential in each compartment AGNa+ = A/d, tNa+o-i-- /[s

/d,tNa+i .

When combined in this way, Eqs. (3.32) and (3.33) simplify to

/d't Na+A ~ /d,tNa+B 9

/.LtNa+A -- fiI,~

(3.17)

q- R T In aNa+B q-- ZNa+ ~ - Xt)'B.

(3.19)

In primary active transport, such movement of solutes is driven by hydrolysis of phosphoric acid anhydride bonds in ATP.

AGNa+ : A/.s = R T l n (aNa+o/aNa+i) nt- ZNa+ ~ - (~Ito -- ~Ifi).

(3.34)

A similar expression can be derived for the total chemical potential gradient of K § across the plasma membrane. In this case, however, the equation is written to reflect the total chemical potential difference against which K § must be moved in order to be taken up by cells. Therefore, the equation for K § transport by Na+K+ATPase is AGK+ = A/d, tK+i_o--

RTln (aK+i/aK+o)

-k- ZK+ ~ (~Iti -- aI*o)

(3.35)

to reflect its movement in the opposite direction of Na + (compare Eqs. (3.34) and (3.35).

50

3. Thermodynamics and Transport

Finally, the thermodynamic expression for intracellular ATP hydrolysis must take into account the reactants and products of the following chemical equation ATP 4- + H 2 0 ~ ADP 3- + Pi 2- + H +

(3.36)

The thermodynamic expression takes the general form of Eq. (3.11) for a chemical reaction A G = AG ~ + R T l n (aB/aA)

(3.11)

AG~

A G = - R T In K e q nt- R T In (as/aA).

Most intracellular biochemical reactions occur, however, in the presence of buffers that maintain the pH value near 7. Moreover, these reactions occur in solutions that are dilute enough in regard to the solutes to allow us to assume that the activity and concentration of the solvent, water, has a large constant value of 55.5 M. Hence, neither the activity of the reactant, H20, nor that of the product, H +, in Eq. (3.36) is usually included in calculations of the values of the observed equilibrium constant, K'eq, the standard free energy change, AG ~ or the total free energy change, A G. While K'eq and AG ~ are marked with a prime to indicate that their values may differ from those that would be obtained when the activities of H20 and H + are included in the calculations, the total free energy change, A G, needs no such designation. If they have not previously done so, readers should satisfy themselves that omission of these activities in the calculation of AG ~ from K ' e q is balanced when the activities of water and protons are also omitted from the general expression, R T ln(aB/aA), thus rendering AG unaltered. Several other factors, such as ionic strength and the activities of metal ions, also influence the free energy of hydrolysis of ATP. One such factor, the Mg 2+ activity, is especially important since Mg 2+ forms complexes with ATP and ADP to form the substrates and products actually involved in intracellular biochemical reactions. The thermodynamic effects of the Mg 2+ activity on several such reactions has been studied in detail (Alberty, 1969). All such activities are, however, also assumed to remain constant for most calculations of the free energy of hydrolysis of ATP. The thermodynamic expression for ATP hydrolysis under physiological conditions can therefore be written d- R T l n

(aADpapi/aATP).

K'eq = [ADPleq

[Pileq/[ATPleq =

2.22 X 105M.

Moreover, AG~ can be calculated from an equation that resembles Eq. (3.8) to be

or when combined with Eq. (3.8)

AGATP = A G ~

the convenient assumption that the values of solute activities are near enough to the values of solute concentrations to allow us to use the latter values in calculations without significant error. Under these assumptions for the ATP phosphohydrolase reaction, K'eq can be calculated from the equilibrium concentrations of ATP, ADP, and Pi to be

(3.37)

Eqs. (3.34), (3.35), and (3.37) can now be used to calculate the free energy changes associated with each of the processes that are coupled bioenergetically by Na+K+ATPase. Since the solutions of solutes both inside and outside cells are relatively dilute, we can also make

= -RTln

g ' e q = - 31.7 kJ mo1-1

at 310~ (R = 8.314 J deg-lmol-1). From this value of AG ~ the actual free energy change at pH 7 and 310~ can be calculated from Eq. (3.37) for any cell in which the cytosolic concentrations of ATP, ADP, and Pi are known. For rat hepatocytes, these concentrations have been estimated to be 3.38, 1.32, and 4.80 mM, respectively. In this case Z~GATP = -31.7 kJ mo1-1

+ R r l n ((0.00132 M)(0.00480 M)/(0.00338 M))

AGATP = -31.7kJ mo1-1 + -16.2 kJ mo1-1 AGATP = -47.9 kJ mo1-1.

More generally, the free energy of hydrolysis under typical intracellular conditions can be calculated to range from about - 4 2 to about - 5 4 kJ mo1-1 (Chow and Forte, 1995). Similarly, Eq. (3.34) can be used to calculate the free energy of Na + extrusion from cells. Typically, the intracellular and extracellular concentrations of Na + are about 11 and 140 mM, respectively (Chow and Forte, 1995), and we are assuming that these concentrations are nearly equivalent to the activities of Na+. Moreover, the membrane electrical potential of cells is frequently about -0.05 V (inside negative). Under these conditions and assumptions AGNa+ = R T l n ([Na+]o/[Na+]i) "q- ZNa+ ~ ('tI'to -- 'tI)'i)

(3.34)

AGNa+ = R T In (0.140 M/0.011 M) -t-9 ZNa+ ~ " (-]-0.05 V )

AGNa+ = 6.56 kJ mo1-1 + 4.82 kJ mo1-1 AGNa+ = 11.4 kJ mo1-1,

where f is 96.5 kJ V -1 equiv -1, R is 8.314 J deg -1 mo1-1, and T is 310~ The stoichiometry of transport is, however, extrusion of 3 Na + and uptake of 2 K + for each ATP hydrolyzed under physiological conditions. Hence, the minimum free energy needed (or the work that must be done) to move 3 moles of Na + out of a cell per mole of ATP hydrolyzed is

Chemical Reactions Drive Primary Active Transport AGNa+T-- 3 X 11.4 kJ(mol of ATP hydrolyzed) -1

51

all types of transport ATPases, including the P-type, V-type, and F-type ATPases (see below and Chapter 5).

AGNa+T = 34.2 kJ(mol of ATP hydrolyzed) -1.

Finally, the minimum free energy needed to move 2 K + into the cell can be calculated using Eq. (3.35) and intracellular and extracellular K + concentrations of 140 and 5 mM, respectively (Chow and Forte, 1995). In this case AGK+ =

R T l n ([K+]i/[K+]o) -Jr- ZK+ ~ (XIri -- xI~ro)

(3.35)

AGK+ = R T l n (0.140 M/0.005 M) -}- ZK+ ~" (--0.05 V )

AGK+ = 8.59 kJ mo1-1 - 4.82 kJ mo1-1 AGK+ = 3.8 kJ mo1-1. Since 2 K § are moved per ATP hydrolyzed, the total free energy needed for uptake of 2 moles of K + per mole of ATP hydrolyzed is AGK+ w =

2 • 3.8 kJ(mol of ATP hydrolyzed) -1

A G K + w --

7.6 kJ(mol of ATP hydrolyzed) -1.

We can now compare in several ways the free energy changes associated with each aspect of the overall process catalyzed by Na+K+ATPase. First, compare the free energy needed to move a mole of K § ions and a mole of Na + ions against their concentration (and activity) gradient across the plasma membrane of a typical cell. Threefold more energy is needed for the movement of Na + in spite of the fact that its concentration gradient is not as steep as that for K § This difference between Na + and K + is, of course, due to the inside negative membrane electrical potential against which Na + must also move, whereas K + is pulled by the inside negative electrical potential against its concentration gradient. In fact, we will show in calculations made in Section X below that K § may in many cases be very near to its equilibrium predicted by the Oibbs-Donnan effect. Hence, we shall see that Na+K+ATPase is frequently more of a Na § pump than a Na+K + pump under physiological conditions. From the above calculations it can also be seen that the total free energy available from ATP hydrolysis (42 to 54 kJ tool -1) is somewhat greater than the energy needed for K § uptake and Na § extrusion (34.2 + 7.6 = 41.8 kJ(mol of ATP hydrolyzed)-l). One may, at first, gain comfort from such calculations performed here and by numerous other authors. Enough free energy to drive the endergonic transport processes is indeed available from the hydrolysis of ATP. The details of coupling of the free energy changes associated with transport and chemical reactions are, however, virtually unknown for

B. C o m p a r i s o n of H o w Well W e U n d e r s t a n d Coupling b e t w e e n Multiple Transport or Multiple Chemical Processes to H o w Well W e U n d e r s t a n d Coupling of Transport to a Chemical Reaction The earlier example of the coupling of movement of Na + with its activity (and concentration) gradient while C1- moved against such a gradient toward the GibbsDonnan equilibrium (difference between Figs. 3.4 and 3.5) makes intuitive sense. It is possible to reason, for example, that an electrical membrane potential develops when Na + begins to move across the semipermeable membrane with its concentration gradient. In this case, C1- also moves in the same direction toward the positive side of the membrane and in so doing creates an activity gradient of this ion. Similarly, we will find that the bioenergetic coupling of protein-mediated transport of two or more solutes frequently seems relatively easy to comprehend (e.g., see Sections IX and X below). We may also assume that we comprehend relatively well the bioenergetics of coupling between many chemical reactions, such as the two that may be viewed as constituting the reaction catalyzed by adenylate kinase ATP 4- + AMp2-

0

0.1

0.2

0.3

0.4

0.5

[S], mM

FIGURE 4.19 Relationship between the concentration of substrate and the velocity of its saturable biomembrane transport. The curve forms a rectangular hyperbola described by the Michaelis-Menten equation (Eq. (4.26)). The horizontal line marked "Vmax"is the maximum velocity approached by the curve at relatively high substrate concentrations. At one-half of Vmax,the substrate concentration is by definition the Km value. In the present case, one-half Vmaxoccurs at a substrate concentration of about 50/xM.

12i =

k2 [MS],

(4.18)

w h e r e vi is the initial velocity, k2 is the rate c o n s t a n t for the step m a r k e d k2 in S c h e m e (4.17), a n d [MS] is the c o n c e n t r a t i o n of the MS c o m p l e x (it is again a s s u m e d that c o n c e n t r a t i o n s can be s u b s t i t u t e d for activities). E v e n t h o u g h a derivation of the Eq. for the h y p e r b o l a s h o w n in Fig. 4.19 (i.e., the M i c h a e l i s - M e n t e n e q u a t i o n ) is p r e s e n t e d in n u m e r o u s b i o c h e m i s t r y t e x t b o o k s , it is

Kinetics of Saturable Transport

also shown below because it can help to provide a foundation for understanding the meaning of the kinetic parameters Km, Vmax, and Ki. At steady state, the rate of formation of MS is equal to the rate of its breakdown. Moreover, these rates take the familiar chemical kinetic forms (4.19)

vf = k l ( [ M T ] -- [ M S ] ) [ S ]

and

85

logical concentrations of substrates, is the Km value (Fig. 4.19). That is, when Km = [S] Eq. (4.26) becomes Vi -- Vma x or Vi ~-~

Vd ~-~

k-1 [MS] + k2 [MS],

(4.20)

when ve and Vd are, respectively, the rates of formation and degradation of the MS complex; kl, kq and k2 are the rate constants for the steps shown in Scheme (4.17); and [MT], [MS], and [S] are the concentrations of the total M present (both substrate bound and unbound), the MS complex, and S, respectively. Since, at steady state

[S]/2[S]

Vmax/2.

For the more general case where more than one of the same ion or molecule may be transported together (e.g., Fig. 4.18), Eq. (4.26) can be modified to read Vi = Vma x [s]n/([S] n .-+- go.5 n)

(4.27)

where n is the number of identical ions or molecules transported together and K0.5 is the substrate concentration at half maximum velocity (Km when n = 1).

Pf = Vd

we may write kl

( [ M T ] - [MS])[S]

= k_ 1

[MS] +

k2

[MS]

(4.21)

Equation (4.21) can be rearranged in several algebraic steps to read

[MS] = [MT][S]/([S] +

( / 2 nu k - 1 ) / k l )

(4.22)

Now the relationships among the three kinetic constants in Eq. (4.22) is defined as the Michaelis-Menten constant, Km K m -- ( k 2 q-- k _ l ) / k I

(4.23)

and Eqs (4.18), (4.22), and (4.23) can be combined to read Vi -- k 2 [ M T ] [ S ] / ( [ S ]

+ Km).

(4.24)

Finally, since the maximum velocity of transport (Vmax) will occur when M is fully saturated (i.e., when [MS] = [MT] Vmax = k2 [ M T ] ,

(4.25)

Eq. (4.24) can be simplified to the familiar form of the Michaelis-Menten equation Vi--

gmax [ S ] / ( [ S ]

+ Kin)

(4.26).

Since the Vmaxvalue for a given process depends only on the total concentration of the catalyst, [MT], vi in Eq. (4.26) approaches the Vmax value as the substrate concentration, IS], is increased above the Km value. Moreover, it can be seen from Eq. (4.26) that the substrate concentration at half-maximal velocity, and the rate discussed in Section VIII above in regard to physio-

B. Fitting of Experimental Data to Kinetic Formulations and Statistical Comparisons of the Values of Kinetic Parameters D e t e r m i n e d from Such Fittings The best fit of transport data to a hyperbola defined by Eq. (4.26) can be determined with nonlinear regression analysis (Atkins and Nimmo, 1980; Gardner and Atkins, 1982; Ritchie and Prvan, 1996). Computer software for performing nonlinear regression analysis is now available commercially from several companies (e.g., Sigma Plot, Jandel Scientific Corp, San Rafael, CA 94901, USA), and the software also can be used to calculate kinetic parameters and to estimate the level of uncertainty in their values. The reader is cautioned, however, that the statistical uncertainties in Km and Vmax values that are determined using nonlinear regression analysis should not be used to compare the values to other such values in statistical tests. Rather, statistically significant differences among the values of Km and Vmax may need to be examined in the familiar way, by obtaining three or more replicate values for the parameters in each of the tissues or conditions under consideration. The mean values and their variances for each tissue or condition may then be calculated and the values compared with appropriate parametric or nonparametric statistical tests. Alternatively, transport experiments that were not designed explicitly to measure Km and Vmaxvalues may nevertheless support the theory that these values are different for different tissues or for the same tissue under different conditions. Such theories may then be tested by obtaining replicate values, as described above, or they can be tested by collecting single sets of kinetic

86

4. Transport Kinetics

data or by combining data into single sets for each tissue or condition. In the latter cases, the method of Eisenthal and Cornish-Bowden (1974), as later modified by these authors (Cornish-Bowden and Eisenthal, 1978), and by Porter and Trager (1977) may be used to compare Km or Vmaxvalues statistically. Briefly, this method involves determining values of Km and Vmaxbased on each pair of data points such as those depicted in Fig. 4.19 and then comparing ranges of values so determined for different tissues or conditions. From such assessments, medians and confidence intervals rather than means and standard errors are obtained. One may then use the finding that the confidence intervals for the values obtained for different tissues or under different conditions either do or do not overlap to conclude that the values are or are not statistically distinguishable from one another at a probability greater than the probability that the intervals contain the true values of the kinetic parameters (e.g., Table 4.1). One may also gain a visual appreciation of whether significant differences may exist between or among the values of kinetic parameters by examining linear transformations of the pertinent data. To produce such graphs, Eq. (4.26) Vi :

Vma x [S]/([S]-Jr- g m )

(4.26)

can be converted algebraically to several forms that represent straight lines for different values of [S] and vi. These three forms of Eq. (4.26) are 1/vi = Km/(Vmax[S]) + 1/Vmax, [S]/v i = [S]/Vma x -Jr- gm/Vma x

(4.28) (4.29)

and

TABLE 4.1 Kinetic Parameters for L-Lysine Transport by M o u s e Blastocysts in Different Isotonic Solutions a

Principal solute(s) in the isotonic solution in which uptake was measured

NaC1 LiC1 Sucrose

Median value of the parameter (92-94% confidence interval) b

Km

Vmax

61(48-90) 59(43-72) 5(1-8)

68(58-75) 42(39-51) 44(32-54)

aA modification (Cornish-Bowden and Eisenthal, 1978; Porter and Trager, 1977) of the nonparametric statistical method of Eisenthal and Cornish-Bowden (1974) was used to estimate the median values of kinetic parameters and their 92-94% confidence intervals (Van Winkle et al., 1990c). When the 92-94% confidence intervals do not overlap, it is more than 95% likely that neighboring values are different because even the 90% confidence intervals overlap somewhat when p = 0.05 in t tests (data from Van Winkle et al., 1990c with permission from Elsevier Science). bKm in/xM and Vmaxin fmol blastocyst -1 min -1.

lei/[S ]

-

-

Vmax/gm-

12i/g m

(4.30)

or

vi = Vmax -- Vi Km/[S].

(4.31)

When data are presented in graphs defined by Eqs. (4.28), (4.29), and (4.30), they are termed, LineweaverBurk, Hanes and Eadie-Hofstee (or simply Hofstee) plots, respectively (Fig. 4.20A-4.20C). The Hofstee plots shown in Fig. 4.21B correspond to Eq. 4.31. One can gain from these linear plots an immediate sense of the magnitude of the differences that may exist among values of Km and Vmax. For example, the differences in the values of the kinetic parameters reported in Table 4.1 are immediately obvious from the linear plots of the data in Fig. 4.21B. The magnitude of these differences may, however, not be as obvious from the nonlinear plots of the data shown in Fig. 4.21A. In Fig. 4.21B, the values of the y-intercepts correspond to Vmax values, whereas the negative values of the slopes correspond to Km values. In contrast, the Vmax values (i.e., the vi values at infinitely high substrate concentrations) are more difficult to estimate from the data as presented in Fig. 4.21A. Similarly, it is difficult to estimate the Km values visually (i.e., the substrate concentrations at onehalf the Vmax values) whenever the Vmax values are, themselves, difficult to judge accurately. While most readers are now familiar with the definitions of Km and Vmax, the relationship of their values to each other and to other kinetic parameters may not be as clear. Moreover, it is useful to consider why transport proteins have circumscribed values for these parameters and how the values may differ for transport in opposite directions across the membrane. For these reasons, we turn now to a more detailed consideration of the meaning of Km and Vmax. C. Meanings of the Kinetic Parameters K m a n d Vmax

1. Finite Values of Km and VmaxAre a Consequence of the Close Biophysical Interactions between Small Ions and Molecules and the Protein Molecules That Catalyze Their Biomembrane Transport

Because biomembranes appear to be barriers to the migration of even highly lipophilic substances, it is possible for the rates of migration of solutes and water across membranes to be increased by catalysts. Even the best catalysts cannot, however, increase the rate of biomembrane transport to near that which would occur if simple diffusion across the membrane were possible. For example, Stein (1986, p. 202) has calculated that gramicidin channels operate at no more than about 8 percent of the rate of migration of K + ions that occur by simple diffusion, and even the fastest K + channel protein mole-

87

Kinetics of Saturable Transport

A

B

C

L

13,/[s] [S]/ar Slope

= KJVM~

Slope

=

9

/

IlVM,~

1

~

SLOPE = - I / K M

VM~ /Ki

l l VMAx - -

-~/K. I

i

~/iS]

_KM

_ _

[S]

Vv~x

~

'

FIGURE 4.20 Lineweaver-Burk (A), Hanes (B), and Eadie-Hofstee (or Hofstee)(C) plots of transport data such as those shown in Fig. 4.19. The plots represent different linear transformations of the Michaelis-Menten equation (Eq. (4.26)) and correspond to Eqs. (4.28) (A), (4.29) (B), and (4.30) (C), respectively (adapted from Stein, 1986, with permission from Academic Press).

cules operate no faster than the channels formed by the gramicidin peptide. Since membranes appear to be barriers to diffusion even when they contain rapid channels, one can expect transport always to be saturable via the pathways through membranes created by integral membrane transport protein molecules. As discussed above, the Km values vary widely among protein cata-

lyzed transport processes. Vmax values also vary widely since their values frequently correlate at least roughly with Km values. These kinetic data also give us insight into how transport protein molecules may function. Since proteins catalyze transport, but the resultant rate never approaches that of free diffusion, even for the fastest transport via

7~

B 60

60

9

I

~

"T,r .n

/////Jl'-''-

I- 5 0 .........=

/

f / f /

E

9

/-

~.~

go

..

9

40 NaCI

m

3 o i / L -/

9

o

sucrose

~ 20

NaCI ........... Sucrose

buffered

LiCI

Phosphate-buffered

LiCI

0 0

i 200

J

400

i

600

[Lysine], I~M

I

800

1000

0

2

4

[L-lysine], ~tM

FIGURE 4.21 Nonlinear (A) and linear (B) plots of L-lysine uptake as a function of lysine concentration by mouse blastocysts under different isotonic conditions (the principal solutes are shown for each line). The lines shown in A represent the best fit for the data points obtained under each condition to a hyperbola, whereas the lines in B represent the best straight lines for the data points after linear transformation (Eq. (4.31)) of the Michaelis-Menten equation (Eq. (4.26)). (B adapted from Van Winkle et al., 1990c, with permission from Elsevier Science).

88

4. Transport Kinetics

channels, transport proteins must interact intimately with their substrates. These intimate interactions may include ionic and hydrophobic interactions and hydrogen bonding depending on the transport process and the chemical structure of its substrate. Such interactions slow the rate at which the substrate moves by free diffusion, even for the short width of biomembranes which otherwise are virtually impermeable to the migration of hydrophilic solutes. An understanding of the details of these close biophysical interactions is emerging for a number of transport proteins (see Chapters 5 to 8 for various examples). We are also only beginning to appreciate the significance of asymmetric insertion of transport protein molecules into biomembranes. Although this asymmetric structural orientation of transport proteins in biomembranes has been known for several years, it is still frequently assumed without much testing that many proteins function symmetrically in regard to their Km and Vmax values. For example, channels are frequently assumed to operate symmetrically in their open states, but their Km and Vmax values for uptake vs exodus have, to our knowledge, not usually been determined and compared. We anticipate that channels would operate symmetrically in regard to their Km and Vmax values about as frequently as do uniporters and antiporters. In this regard, nonaccumulating glucose transport has been shown to be directionally symmetric (i.e., Km ex~ -- Km uptake, Vmaxex~ = Vmaxuptake) in rabbit erythrocytes (Regen and Morgan, 1964) and rat hepatocytes (Craik and Elliot, 1979), but asymmetric in human erythrocytes (Wilbrandt, 1955; Bloch, 1974) and rat thymocytes (Whitesell et al., 1977). In our view, symmetric operation of transport proteins should be viewed as coincidental or possibly due to evolutionary selection for such function, since the precise mechanisms of transport are unlikely to be symmetric for asymmetrically oriented protein molecules in biomembranes. Obligatory exchange or antiport might, at first, seem to be an exception to this conclusion. A moments reflection should, however, lead us to drop that exception. Even for exchange, the values of the kinetic parameters need not be identical for uptake and exodus. Such has in fact been found to be the case for nonobligatory glucose exchange (Bloch, 1974) and obligatory anion exchange (Chapter 6), although the exchange itself is, by definition, symmetric. Ironically, many enzymes for physiologically reversible reactions may operate in a more symmetric manner than do transport proteins, even though enzymes catalyze chemical changes, whereas transporters do not destabilize their substrates. For example, amino transferase reactions are catalyzed by proteins with more or

less invariant sites for binding of amino acids and 2-oxo carboxylic acids (c~-keto carboxylic acids) regardless of whether they catalyze the forward or the reverse of reactions of this type

~C

_

R1

+NH 3

I

COO- + H m C m C O O -

I

R2 +NH3

- H m CI ~ C O O - +

I

R1

(4.32) O

'

I

c-coo

R2

In the above reaction, the enzyme molecule is structurally about the same whether it receives the substrates on the left or the substrates on the right. In contrast, a transport protein molecule presents a different orientation to identical substrate molecules on opposite sides of a biomembrane. For this reason, the effectiveness of transport proteins as catalysts, and hence their Km and Vma x values, could vary considerably for migration of substrates in one direction across biomembranes vs the other direction. Asymmetric transport should not be confused with an asymmetric biochemical reaction. A biochemical reaction may be asymmetric because the structures of the reactants and products are quite different, not because the enzyme catalyst is different for the forward and reverse reactions. Moreover, although the enzyme may receive substrates and products for the forward and reverse reactions differently, it treats identical substrate molecules in the same way. In contrast, identical substrate molecules on opposite sides of a biomembrane are received differently by the same transport protein molecule. The possibility that virtually all transport proteins exhibit structural and hence functional asymmetry for identical substrate molecules on opposite sides of biomembranes may have heretofore unappreciated thermodynamic as well as kinetic implications.

2. Can Transport Proteins Produce Solute Gradients Owing to Their Asymmetric Structures and Functions and the Free Energy Needed to Produce and Maintain Such Asymmetries? It has been concluded elsewhere that transport that is not coupled to a conspicuous source of free energy in ATP or a solute gradient may nevertheless have different values of the kinetic parameters for uptake and exodus across the plasma membrane without breaking the laws of thermodynamics as long as the ratios of the Km to Vmax values for uptake and exodus

Kinetics of Saturable Transport

are equal. Just such equal values for the ratios of these kinetic parameters have in fact been found in many studies. It is also conceivable, however, that the ratios of Km to Vmax as well as their absolute values need not be identical for uptake and exodus. Such transport should produce total chemical potential gradients, which for substrates with no net charge can be expressed entirely as gradients of activity or concentration. Although the sources of free energy needed to drive such transport processes remain to be identified, the processes could conceivably be driven simply by the free energy needed to construct and maintain asymmetric biomembranes. 5 In addition to their asymmetric orientations in biomembranes, most transport protein molecules are believed to undergo conformational changes in order to be able to receive substrates for transport on one side of the membrane or the other. While the differences in the free energies of the different conformations of transport proteins have not been measured in most instances, these differences in free energy would not need to be large to produce modest solute gradients. For example, a &G value for the different conformations of 1.8 kJ mo1-1 would be needed to maintain a twofold greater steady state concentration of an uncharged solute on one side of a biomembrane than on the other (see Eq. (3.34) and several similar equations in Chapter 3). Moreover, different conformations of allosteric proteins differ in their free energies by an average of about 22 kJ mo1-1 (Goldsmith, 1996). Hence, the free energies of different conformations of a transport protein would need to differ by less than 10% of the average such difference for allosteric proteins in order to produce a twofold difference in solute concentration across a biomembrane. In this regard, the affinity constants of various inhibitors (measured directly) or substrates (measured indirectly) for binding to the two conformations of the anion exchange protein, band 3 (Knauf et aL, 1992), can be used to calculate a free energy difference (Goldsmith, 1996) of about 3.4 to 6.0 kJ mol -~ for the two conformations, depending on the substance bound. It remains to be determined, however, whether band 3 or other transport proteins are themselves inherently able to produce gradients in the total chemical potentials of solutes across biomembranes (see also Chapter 6). 5 The proposed need for continuous free energy input to maintain constituents of biomembranes in asymmetric orientations warrants emphasis here. If free energy were not expended to maintain membranes and their constituents they would soon stop performing their functions. Maintenance of transport proteins in asymmetric conformations may be viewed as gradients that could conceivably be converted into solute gradients. Both gradients would be maintained only through a more or less continuous free energy expenditure by cells. Asymmetrically oriented transport proteins and their maintenance are, therefore, different from Maxwell's demon or a perpetual motion machine of the second kind.

89

It is, nevertheless, possible to imagine how the free energies of the different conformations of uniporters, symporters, and antiporters might be utilized to produce total chemical potential gradients of their substrates across biomembranes. These possible mechanisms for the production of gradients by the transport protein per se are described here only to demonstrate the conceptual feasibility of such processes. In the case of a uniporter, one conformation of the transport protein might be favored when substrate is bound, while the other is favored in the unbound state. A similar mechanism could produce gradients of two or more solutes via symport, while antiporters could produce gradients through obligatory exchange of two different solutes each of which favors a different conformation of the transport protein molecule. On a macroscopic level, structures with functions analogous to those proposed for asymmetrically organized transport proteins are easy to imagine. Cages or traps with doors that open in only one direction are an obvious example of how a barrier with an asymmetrically constructed entrance can provide greater concentrations of animals on one side of the barrier than on the other. In this case, the asymmetric door influences the thermodynamics as well as the kinetics of migration in one direction relative to the other. The asymmetric door also may be viewed as having different conformations when viewed from different sides of the barrier. The asymmetric distribution of these conformations across the barrier forms a gradient that can be propagated into a gradient of animals. The overall process is exergonic, owing to the conversion of the free energy expanded to construct the device into the free energy of the device itself. A continued input of free energy would also be required to repair the device and thus prevent the intended function of the device from deteriorating. In our view, current dogma sometimes equating migration of solutes through channels or uniporters to diffusion has militated against the design of studies to determine whether some of these proteins can serve to produce total chemical potential gradients of solutes or even the solvent. Similarly, the covert assumption most of us make in assessing the thermodynamics of transport entirely as a function of solute total chemical potential gradients (e.g., as we did in Chapter 3) is that transport protein molecules operate in a thermodynamically symmetric manner in the absence of coupling to an obvious source of free energy in ATP or in a previously formed solute gradient. Now that we know that the structures of most if not all transport protein molecules are asymmetric in biomembranes, however, we need to consider the possible thermody-

90

4. Transport Kinetics

namic as well as kinetic consequences of their asymmetric operation. 6 In this regard, we must also consider the converse. That is, some of the membrane-spanning helices of transport protein molecules may migrate within or even into and out of the lipid bilayer. The extreme (and we think unlikely) consequence of such migrations would be the complete reversal of the orientation of components of the protein molecule within the membrane. Transport protein molecules might lose asymmetry to the extent that they reverse their orientations in biomembranes. Investigation of these possibilities will, of course, require further study of the relationships between the structures of transport proteins and the values of their kinetic parameters for transport of substrates in both directions across biomembranes. It should also be useful to understand the relationship of the values of these kinetic parameters to the affinity (or dissociation) constants, since the latter values may be used to determine the differences in free energy of different conformations of transport protein molecules. Toward these ends, the structures and functions of several transport related proteins are considered in greater detail in Chapters 5 to 7.

3. Relationship of Km to the Substrate Dissociation Constant (Kd) and to Vmax Several additional interrelated concepts concerning the meanings of the Km and Vmax values also warrant consideration. Most of these concepts are intended to apply best to the simplest type of transport via channels or uniporters, although the concepts also may apply reasonably well to aspects of more complex types of transport. First, it is frequently and incorrectly asserted, even in the current literature, that the gm value is virtually equivalent to the dissociation constant (Kd) or to the inverse of the affinity of a substrate for its transporter. Such is the case, however, only in relatively rare instances where an equilibrium as well as a steady state assumption can be made in regard to Scheme (4.17). Since k2 usually contributes significantly to the value of Km in the simple representation of transport in Scheme (4.17), the Km value (Eq. 4.23) will also usually exceed the Kd value (Kd = k-Jka) when this Scheme applies. 6 While our discussion here has been primarily a theoretical one, we present evidence in Chapter 7 (Section II,C) that inwardly rectifying K + channels may indeed catalyze the migration of K + ions against their total chemical potential gradient. Such studies have, however, not been performed intentionally to test the present hypothesis that transport proteins may catalyze such migration owing to their asymmetric orientations in structurally asymmetric membranes. Experiments designed with the intent of determining whether channels and uniporters may sometime transport solutes against their total chemical potential gradients are, therefore, needed to test our hypothesis.

Historically, the fact that the Km value usually differs from the Kd value has been important in kinetic assessment of whether a transport process operates symmetrically or asymmetrically. In particular, the mobile carried model implied that the transport mediator might present the same face to substrates on either side of the membrane in some instances of passive transport. Hence, the value of Kd was expected to be the same at the inside and outside surfaces of the membrane, even when the transport process operated asymmetrically. In contrast, the Km values for uptake and exodus were expected to differ for asymmetric transport. As discussed in Section 2 above, it has until now been expected for asymmetric transport that the ratios of the Km to Vmax values would be equal for uptake and exodus via transport processes that are not coupled to an immediate source of free energy. Although it may now be anticipated that the values of these ratios need not be identical, we still expect a correlation between Km and Vmax values because the Km value is at least partially dependent on the values of rate constants that also help to determine the Vmax value. The Km and Vmax values should thus be viewed as measuring some of the same aspects of transport. For example, in the simplest formulations for transport under the steady state approximation, both the Vmax value (Eq. (4.25)) and the Km value (Eq. (4.23)) depend directly on the value of k2. It is therefore not surprising that the Km and Vmax values are correlated; transport processes with higher Km values also frequently have higher Vmaxvalues than processes with lower Km values. Nevertheless, the ratios of Km t o Vmax need not be precisely the same for exodus and uptake, owing to the asymmetric conformations of transport proteins in biomembranes. Now, as discussed above, in the simplest formulation for transport (Scheme (4.17)) Km~Kd because (4.23)

Km = (k-1 -k- k2)/kl, whereas

Kd = k-1/kl. For multistep transport models, however, the Km value may be either larger or smaller than Ko. For processes in which only one more step than in Scheme (4.17) is inserted kl ~ k3 S~ + M ~ (MS)I (MS2) ~ M + k -1 -2 the Km value can be shown to be

S2

(4.33)

Kinetics of Saturable Transport

Km = (k_flk~)(k3 + k-2 + k2(k3/k-1))/ (k3 + k-2 + k2),

(4.34)

where lowercase k's are the rate constants shown in Scheme (4.33). It can be seen from Eq. (4.34) that whether the Km value is larger or smaller than Ka depends on the ratio of the rate constants for the exit of S from the transporter on each side of the membrane. A procedure for deriving the expression for the Km value in Scheme (4.33) and in more complex cases is given in Appendix A of Stein (1986). Scheme (4.33) may apply best to channels, whereas additional considerations involving the reorientation of the protein to its original position without its substrate have historically been applied to carriers such as uniporters under zero trans conditions. In this regard, it should be noted that only modest success has been achieved in determining whether actual transport processes fit kinetically more complex models of transport, such as the somewhat more complex one shown in Scheme (4.33), or whether they fit the simple model in Scheme (4.17). Modern molecular studies indicate that many types of transport involve multiple steps (Chapters 5 to 7) which display only simple carrier kinetics (Hern~indez, 1998). Apparently these multiple steps either are not detected well by current kinetic procedures or they are not modeled well by known kinetic formulations (but see also Chapter 11). Finally, for the kinetically more complex process of symport, the relative values of Km and Vmax for one substrate at various concentrations of the other can, in theory, be used to determine the order of substrate binding. For example, when one substrate molecule must bind first, changes in its concentration are not expected to influence the Vmax value of the other substrate (Stein, 1986). While one may be inclined to question a kinetic model that makes such a counterintuitive prediction, several instances where the concentration of one substrate does not influence the Vmax value of the other substrate have been reported in the literature (summarized in Stein, 1986). The latter data could, of course, also result from undetected uncoupling or slippage of the cotransport process (i.e., its reversion to uniport of a single solute). Moreover, it has been concluded in more recent studies that one substrate molecule binds before the other even when a decrease in the concentration of the first substrate is observed to decrease the Vmax value of the other (e.g.; Boorer et al., 1996; Mackenzie et al., 1996a). For such reasons it is somewhat difficult to judge whether any currently emerging kinetic model of cotransport accurately reflects accumulating knowledge of the molecular structures and functions of symporters (see Chapter 6 for further discussion of the difficulties associated with studying the effects

91

of cosubstrates on each other's transport). Current procedures for studying the details of the kinetics of transport and the theoretical considerations needed to interpret the results of such experiments are discussed more thoroughly in Chapter 11. D. The Parameter, K~ in Inhibition Analysis 1. Determination of K~ Values The Ki value of a competitive inhibitor of a transport process frequently is equal to the Km value for transport of the inhibitor by the same process. For this reason, we shall see that determination of Ki values can be particularly useful in testing whether two or more solutes share a transport process (Section 3 below). Historically, competitive inhibitors are viewed as raising the Km value of a substrate without altering its Vmax value, whereas noncompetitive inhibitors decrease the Vmaxvalue of a substrate without changing its Km value. In Hofstee plots (Eq. (4.31)) competitive inhibition is reflected by an increase in the magnitude of the slope of the line defined by the data points (recall that slope = -Km), whereas noncompetitive inhibition results in a decrease in the y-intercept (Vmax value) (Fig. 4.22A). In practice, more complex types of inhibition are also observed in which both the Vmaxand Km values are affected (e.g., arginine inhibition of taurine transport shown in Fig. 4.22B). These other types of inhibition have been discussed extensively by other authors (e.g., Dixon and Webb, 1964). In cases of competitive inhibition, such as fl-alanine inhibition of taurine transport (Fig. 4.22B), the Ki value can be calculated from the equation. Ki = [I]/((Kmapp/Km)- 1),

(4.35)

where [I] is the inhibitor concentration, Km has its usual meaning, and gmapp is the apparent value of gm for substrate transport in the presence of the inhibitor. A more direct graphical method with which to determine the Ki value and whether inhibition is competitive or noncompetitive is to determine vi at multiple concentrations of both the inhibitor (I) and the substrate (S). When the resultant data are assessed in a Dixon plot (i.e., a plot of 1/vi vs the inhibitor concentration at several concentrations of the substrate) the resultant series of straight lines for different concentrations of the substrate intersect at a point where the inhibitor concentration on the x-axis is equal to the negative of the Ki value. Moreover, such lines intersect above the x-axis for competitive inhibition (Fig. 4.23A), whereas they intersect on the x-axis for noncompetitive inhibition (Fig. 4.23B). These results can be explained by considering that

92

4. Transport Kinetics

B

A

150 0

,_\,

I .o,n.,b,.o.

- \~ Competitive~ '\ In~bition ~

xr/

125

100

~

5O

9

?n

hibitor

./

Noncompetitive~

"~

~:

r \ I

\

%

25

%

\ \

I

%

9

I

lOmM . \

0

~.

, 0

2

a 4

I 6

/[Taurine], nl-conceptus -~. h -~

FIGURE 4.22 Competitive and noncompetitive inhibitors of transport produce decreases in the slope (more negative) and y-intercept, respectively, of Hofstee plots. (A) Changes in the Km (negative of the slope) and Vmax (y-intercept) values of transport owing to competitive and noncompetitive inhibition, respectively, as determined using Hofstee plots (Eq. (4.31)). (B) Inhibition of taurine transport by/3-alanine is competitive, whereas inhibition by L-arginine appears to be mixed (i.e., both competitive and noncompetitive) (B adapted from Van Winkle et al., 1994, with permission from Elsevier Science).

w h e n t h e s u b s t r a t e c o m p e t e s with t h e i n h i b i t o r , t h e inh i b i t o r s h o u l d h a v e no effect at infinite s u b s t r a t e conc e n t r a t i o n . H e n c e , t h e line at infinite s u b s t r a t e c o n c e n t r a t i o n m u s t be p a r a l l e l to y e t a b o v e t h e x-axis if t h e

A

s u b s t r a t e is to h a v e a finite Vmax v a l u e (Fig. 4.23A). In c o n t r a s t , n o n c o m p e t i t i v e i n h i b i t o r s a r e e x p e c t e d to slow t r a n s p o r t , e v e n at infinite s u b s t r a t e c o n c e n t r a t i o n (Fig. 4.23B).

B

=.

;i"

J,,

[i]

_

[I]

FIGURE 4.23 Dixon plots for competitive (A) and noncompetitive (B) inhibition of biomembrane transport. Competitive inhibitors become increasingly less effective as the substrate concentration is increased (A). In contrast, noncompetitive inhibitors are equally effective regardless of substrate concentration (B). The negative of the value of x at the point at which the lines intersect is equal to the Ki value.

Kinetics of Saturable Transport

2. The K~ Values for Competitive Inhibition of Transport Frequently Have a Different Meaning Than for Inhibition of Enzyme Catalysis Competitive enzyme inhibitors bind reversibly to enzymes, and they compete with the substrate for such binding. Competitive inhibitors of enzymes are, however, frequently not also substrates of the enzyme. In such cases, the Ki values for these inhibitors are equal to the values of their dissociation constants (Ka values; Fig. 4.24). In contrast, competitive inhibitors of catalysis by transport proteins usually vie with the substrate both for binding and for transport. For this reason, the Ki value of a substance for competitive inhibition of transport is usually identical to its Km value for transport (Fig. 4.25). For example, the Km values for transport of Lalanine, L-lysine, and the bicyclic amino acid analog 3-aminoendobicyclo [3,2,1 ] oct ane- 3- carb oxylic acid (BCO) are (within experimental variability) identical to their Ki values for inhibition of transport of each other via amino acid transport system B ~ (see Section 3 below). In some instances, however, competitive inhibitors may not be transported. For example, D-tryptophan competitively inhibits L-tryptophan transport by amino acid transport system T (L6pez-Burillo et al., 1985; Van Winkle et al., 1990b), but o-tryptophan does not appear to be a system T substrate. Similarly, cationic amino acids are competitive inhibitors of amino acid transport system ASC, but they are not transported by it (Thomas and Christensen, 1970). Moreover, maltose and phlorizin are reversible competitive inhibitors of Na+independent glucose uptake by G L U T transport proteins, but these substances are not transported by the proteins (Baldwin, 1993). In these cases, inhibition corresponds to the simpler Scheme where Ki = Kd (Fig. 4.24) rather than where Ki = Km (Fig. 4.25). Because it is possible, albeit infrequent, that competitive inhibitors of transport may not themselves be transported,

kI E +S

=

k. 1

k2 >

ES

> E +P

4-

I

k -1

1

K.= n

p

k'

-1

1

-K'

d

El

FIGURE 4.24 Competitive inhibitors of enzyme action frequently do not serve as enzyme substrates. In such c a s e s Ki values are equal to their dissociation constants (Kd values).

93 kI

M+S 1

,i

+

k2 MS

M+S 2

k. 1

11

MI

k'.l + k' 2 k"2

K. ~

n

~

~

,

kl

K

m (I)

M

FIGURE4.25 Competitiveinhibitors of biomembrane transport are also usually substrates for transport. Hence their Ki values are usually equal to their Km values for transport.

considerable evidence must be obtained to support the conclusion that a competitive inhibitor is transported by the same transport process that it inhibits. In part because Ki = Km when the inhibitor is also a substrate, measurement of Ki values is of considerable importance in the inductive process, known as A B C testing (see below), in which an investigator attempts to determine whether two or more solutes share a transport process. 7

3. Importance of Quantitative Inhibition Analysis in Determining Whether Two or More Solutes Compete for the Same Transport Process When two substances mutually inhibit each other's transport, it is occasionally and erroneously concluded that they share a transport process. Such conclusions may arise in part from the converse and correct conclusion that if two solutes do not inhibit each other's transport, then they do not share a transport process. When inhibition does occur, however, it may be noncompetitive, and even mutually competitive inhibitors are not necessarily substrates for the same transport process (see above). In order to demonstrate competition of 7A different procedure that does not rely on the values of these kinetic parameters may be used to characterize other types of transporters. For example, in the cases of channels, substrate-saturable transport may be more difficult to study or a given transporter may have only one known substrate. In many such cases, it has been possible to identify high-affinity inhibitors that act more or less selectively on particular transport proteins or sets of proteins (see summaries and consideration of such inhibitors of channels in Hille, 1992).

94

4. Transport Kinetics

two solutes for the same transport process, evidence must be gathered to show that each solute behaves as an inhibitor in the same way that it behaves as a substrate. 8 W h e n mutual inhibition of transport is observed between two or more solutes, it is first necessary to determine whether the inhibition is competitive or noncompetitive. In the case of Na+-dependent transport of L-alanine, L-lysine, and B C O by mouse blastocysts (system B ~ in Table 4.2), these determinations were m a d e using Dixon plots (e.g., Fig. 4.26). Since these amino acids inhibited each other's transport competitively, the Ki values d e t e r m i n e d from the Dixon plots could be used further to test the hypothesis that these three substances share the same transport process. In Section 2 above it was learned that when an inhibitor is also a substrate of a transport process, its Km value for transport is equal to its Ki value for inhibition of transport of another substrate. For the present example, since the Km value for L-alanine transport is the same as its Ki value for inhibition of B C O transport, and since the Km value for B C O transport is equal to its Ki value for inhibition of L-alanine transport (Table 4.3), these two substances appear to share the same transport process (AB portion of the A B C test). If either equivalency had not been observed, then it could be concluded that different unshared processes (or possibly multiple shared processes) are responsible for transport of each of the two substances (see Section X below for further discussion of such heterogeneity). Even if the values of Km and Ki for a given solute are the same, however, they may be equal coincidentally rather than because the solute interacts with the same process as an inhibitor and as a substrate. For this reason, it is frequently desirable to seek additional evidence that two solutes share a transport process. Such evidence can be gained by studying quantitatively the effects of additional, potential inhibitors of transport of the substrates thought to share a transport process. If the additional solutes are found to have the same Ki values for the competitive inhibition of transport of each putative substrate, then it becomes more likely that the substrates share a transport process (C portion of the A B C test). If, however, any of the additional solutes affects transport of putative substrates differently, then either the substrates do not share the same transport system or multiple shared or unshared 8 We cite amino acid transport experiments especially beginning at this point in the text principally because the potential for using the values of kinetic parameters to show competition for transport via the same agency (systems summarized in Table 4.2) is greatest among the amino acids (see discussion in later Sections). Examples of transport catalyzed by agencies with substrate selectivities other than amino acids are discussed in detail in Chapters 5 to 7. The author asks the readers indulgence for his selecting his own work for purposes of illustration rather than many fine studies of other investigators.

TABLE 4.2 Summary of Some of the Distinguishing Characteristics of Amino Acid Transport Systems a Na+-Dependent systems A. For zwitterionic amino acids System A: prefers less bulky substrates System ASC: can be distinguished from system A by lack of reactivity with N-methyl substrates System B: broad scope system in trophectoderm of mouse blastocysts and possibly other epithelia System B~ A system in renal and intestinal epithelia that may be identical to system B System Gly: has strong preference for glycine and sarcosine System N: prefers glutamine, histidine, and asparagine B. For cationic and zwitterionic and amino acids System B~ broad scope system in oocytes, early conceptuses, and possibly some adult tissues C. For anionic amino acids System X-AG: prefers glutamate, aspartate, and other relatively small anionic substrates II. Na+-Independent systems A. For zwitterionic amino acids System asc: appears to be analog of system ASC System L: prefers bulky substrates System T: selects for benzenoid substrates B. For cationic and zwitterionic amino acids System b~ prefers large substrates that do not branch at the a- or/3-positions C. For cationic amino acids System bx+: substrate selectively nearly limited to cationic substrates System b2+: similar to bl + but interacts differently with specific substrates System y+: prefers cationic substrates but also transports certain zwitterionic substrates with Na + System y+L: higher affinity than prototypic system y+ for bulky zwitterionic substrates in the presence of Na + (distinct for system L, which is Na + independent for transport of zwitterionic substrates) D. For anionic amino acids System Xc-: prefers glutamate and relatively large anionic substrates

I.

aAdapted from Van Winkle, 1993, with permission from Elsevier Science.

transport processes are present. In the case of Na +d e p e n d e n t amino acid transport by blastocysts, L-valine and 2-aminoendobicyclo(2,2,1)heptane-2-carboxylic acid ( B C H ) inhibit transport of both L-alanine and B C O competitively with the same K / v a l u e s (Table 4.3), although the Ki values of L-valine are, of course, not expected to equal those of B C H . These data were used to support the conclusion that a single Na+-dependent system, t e r m e d B ~ transports both L-alanine and B C O in mouse blastocysts. The results of A B testing b e t w e e n L-alanine and L-lysine indicate that L-lysine also is probably a substrate of system B ~ The r e a d e r may observe, however, that more data are presented in Table 4.3 to

Kinetics of Saturable Transport

A

0.40

B

95

0.20

.c_

E 6.4 ~

o .Q

L-alanine

0.20

6.4 pM L-alanine

0.10

o

E T--

16.4 pM L-alanine

16.4 I~M L-alanine

46.4 I~M L-alanine -1.25

2.50

5.00

[BCO] (mi)

46.4 ~M L-alanine -0.15

0.30

[L-lysine] (mM)

0.60

FIGURE 4.26 Dixon plots of BCO (A) and L-lysine (B) inhibition of Na+-dependent L-alanine transport by mouse blastocysts. Inhibition is competitive in both cases as indicated by intersection of the lines above the x-axis (see also Fig. 4.23). The process catalyzing the transport depicted was subsequently designated system B~ (adapted from Van Winkle et al., 1985, with permission from American Society for Biochemistry & Molecular Biology).

support the conclusion that B C O and L-alanine share the same system than that L-lysine and L-alanine do. Moreover, B C O and L-lysine are thought to share system B ~ because they both compete for transport with L-alanine. Mutual inhibition of transport b e t w e e n B C O and L-lysine was not studied quantitatively. Hence,

TABLE 4.3 ABC Testing Indicates That L-Alanine and BCO Share a Na+-Dependent Transport System in Mouse Blastocystsa

Inhibitor and Ki (or Kin) b value (pM) Substrate BCO L-Alanine L-Lysine

BCO 430 320 --

L-Alanine BCH 29 35 35

1000 990

L-Valine L-Lysine 110 101 m

110 140

aln addition, the AB portion of the ABC test indicates that L-alanine and L-lysine share the same system. That is, the Km and Ki values in each column for BCO, L-alanine, and L-lysine (AB portions of ABC tests) and the Ki values in each column for BCH and L-valine (C portion of the ABC test) are statistically indistinguishable from each other based on the known experimental variability in such values for blastocysts (Van Winkle et al., 1990a-c). Hence, it can be concluded that L-alanine, BCO, and L-lysine are all transported by the same Na+-dependent system in blastocysts (see text for details). This system is termed B~ (data from Van Winkle et al., 1985, with permission from American Society for Biochemistry & Molecular Biology). bKm values are reported when an amino acid is listed as its own "inhibitor."

when concluding that two or more substances share the same transport process, both deductive and inductive processes may be particularly evident. Such conclusions may gain additional support over time, since the potential for A B C testing is often much greater than the testing that is actually performed.

4. Further Importance of Understanding the Meanings of Ki and Km When Designing Experiments Involving Inhibition of Transport A serious conceptual error in experimental design still appears occasionally in the current literature. In such studies investigators assume that by using inhibitor concentrations 10- or lO0-fold above the substrate concentration, they will detect inhibition by solutes that compete with the substrate for transport. Fortunately, since Km and Ki values frequently fall in the range of concentrations of substrates and inhibitors used in such studies, the anticipated inhibition is often detected despite the inattention to experimental design. I m p o r t a n t inhibition may, however, go undetected if such experiments are designed without preliminary recognition of approximate Km and Ki values. This point can be m a d e more obvious by considering a simple hypothetical example. Let us assume that an investigator plans to study previously u n e x a m i n e d amino acid transport processes in one of n u m e r o u s cell types that need to take up arginine. The investigator

96

4. Transport Kinetics

selects 10/xM L-arginine as the substrate concentration for initial screening and decides to test a series of amino acids and amino acid analogs as possible competitive inhibitors of the transport process. The investigator reasons that since the substrate concentration is 10 txM, he will be able to detect inhibitors that may also be good substrates by setting the inhibitor concentrations at 100 txM. The experimental design will work very well indeed if the Km and Ki values are all near, say, 10 tzM. If, however, the Km and Ki values are near 500 tzM for the best substrates and inhibitors, then only about 16% inhibition is anticipated, and this amount of inhibition may not be found to be statistically significant. For a transport process with Km and Ki values near 500 p.M, the process will, of course, appear not to be substrate or inhibitor saturable if the concentrations of substrate and competitive inhibitors selected for study are well below 500/xM. Before beginning such inhibition studies, investigators should obtain estimates of Km and Ki values. Alternatively, relatively low substrate and high inhibitor concentrations may be selected based on knowledge of the range of Km and Ki values for similar transport processes. For amino acid transport, a substrate concentration of i /zM is below the Km (and Ki) values of substrates for most known systems, whereas inhibitor concentrations of 10 mM exceed most such Ki (and Km) values. Since the substrate concentration is probably below the Km value for its transport, while the inhibitor concentrations are most likely above their Ki values for inhibition of transport, studies designed in this manner should reveal the anticipated inhibition. It is also important to note that these studies are made possible by the high purity of most commercially available preparations of individual amino acids (or on our ability to purify them before use in experiments), at least in regard to contamination by other amino acids. Nevertheless, some contamination by substances that inhibit transport may occasionally be encountered. Moreover, some cationic or anionic amino acid analogs at 10 mM may be deprotonated or protonated at neutral pH to an extent large enough also to interact significantly with systems for zwitterionic amino acids that have Km values near 1/xM. For these reasons, investigators must be willing to repeat such inhibition studies with different substrate and inhibitor concentrations when more precise determinations of some Km and Ki values indicate that the results of the initial study may not accurately represent the characteristics of the transport system(s) present.

5. Possible Significance of Other Types of Inhibition Our focus in this section has been principally on competitive inhibition and the meaning of Ki (and Km) values. We noted above, however, that inhibition may be

complex as indicated by the mixed (both competitive and noncompetitive?) inhibition of taurine uptake by arginine (Fig. 4.22B). In a different example, inorganic and organic cations inhibit cationic amino acid transport via system b ~ competitively (e.g., Fig. 4.21B), but incompletely (e.g., Fig. 4.27). In this case, the Ki values for Na + and Li + can be calculated to be about 14 mM (see Fig. 4.21B and Eq. (4.35)) but these calculations obscure the fact that inhibition by the cations is inhomogeneous. For example, using the Ki value of 14 mM for monovalent cations, it can be calculated that the rate of lysine transport is somewhat slower than anticipated at 18.8 mM Na + (sodium acetate in Fig. 4.27), whereas it is nearly 70% more rapid than anticipated at 150 mM Na +. The latter data for the acetate salt of sodium can not be explained by activation of Na+-dependent transport at the higher Na § concentration because Na § dependent amino acid transport also requires C1- ions in blastocysts (Van Winkle et al., 1988b) and because the same effect is observed for other cations (Figs. 4.21B and 4.27). Furthermore, cations per se do not influence leucine transport by system b ~247Apparently, cations influence transport via system b ~ at a cation receptor subsite for the side chains of cationic amino acids. The substrate receptor site does not appear to include this cationspecific subsite when zwitterionic amino acids are transported. The resistance of lysine transport to complete

TMAO (I-I) NaCI (0) Taurine (A) Na Acetate (O) Choline CI ( I )

A

"7 C: ~

~E c~

=.o -i"

9

E 0

0 I 300

I

.....

milliOsmolarity I, , milliOsmolarity

I,,

150 of indicated I 150

I

substance I

I

3o0 i

o

of sucrose

FIGURE 4 . 2 7 Effect of cationic substances on L-lysine uptake via system b ~ When the milliosmolarity of the indicated substances was less than 300, sucrose was used to adjust the total milliosmolarity to this value. Inhibition can be shown to be attributable to cations rather than anions as described elsewhere (adapted from Van Winkle et al., 1990c, with permission from Elsevier Science).

Kinetics of Saturable Transport

inhibition by inorganic and organic cations (Fig. 4.27) may therefore occur because the lysine receptor site includes both the distinct cation receptor subsite and a part of the site that receives zwitterionic amino acids. Cations that are not a-amino acids would not compete with lysine for the latter site and, hence, may not be able completely to inhibit lysine transport (Fig. 4.27). It has been proposed that similar sites and subsites may also be present on proteins responsible for amino acid transport via systems y+, ASC, and asc and that these receptor sites or subsites may, therefore, have a common evolutionary origin (Van Winkle et aL, 1990c). The cation harmaline is an especially strong competitive inhibitor of cationic amino acid transport via systems b ~ asc, and y+, and it competitively inhibits the interaction of Na + with the Na+-dependent system ASC (Young et aL, 1988, 1991; Van Winkle et al., 1990c). Moreover, the Ki values for harmaline inhibition of systems b ~ and y+ are considerably higher in isotonic solutions of electrolytes than in solutions of nonelectrolytes. Hence, harmaline appears to interact with the putative cation receptor subsite described above. Harmaline is, however, also a noncompetitive inhibitor of zwitterionic amino acid transport via systems b ~ asc, and ASC. If Scheme (4.17) applies to these transport systems, then harmaline probably decreases the value of the rate constant k2 for zwitterionic substrates, whereas it most likely increases k_l/ka for cationic amino acids. Such an effect on k-1/kl could be due largely to competition between harmaline and cationic amino acids for binding at the putative cation receptor subsite. Selective reduction of the k2 value for zwitterionic but apparently not for cationic amino acids may also result from interaction of harmaline with the same subsite as cationic amino acids. Presumably, when a cationic amino acid molecule occupies the cation receptor subsite, harmaline cannot influence the value of k2 because harmaline is not bound to the subsite. In the case of Na+-dependent transport of zwitterionic amino acids via system ASC, harmaline competes with Na + for interaction with the putative cation receptor subsite. Thus it is likely to reduce the k2 value and, hence, vi, regardless of the concentration of the zwitterionic amino acid substrate. Similarly, zwitterionic amino acid transport via system y+ is Na+-dependent, although the effect of harmaline on transport has not been investigated in that case. Nevertheless, one might predict from these data that an as-yet unidentified cation is required for symport or antiport of zwitterionic amino acids via systems b ~ and ascl. Harmaline could produce noncompetitive inhibition of zwitterionic amino acid transport by competing with such a cation for its receptor subsite. In fact, it has been suggested that uptake of zwitterionic amino acids via system b ~ occurs by obliga-

97

tory exchange for intracellular K + or amino acids, some of which are cationic (see Section XI,E below). If harmaline is taken up by cells, then it could competitively inhibit exodus of cationic substrates and, hence, act as a noncompetitive inhibitor of zwitterionic amino acid uptake via system b ~ Alternatively, when harmaline is bound to cation receptor subsites, it may be bulky enough partially to obstruct the pathway through the membrane for zwitterionic amino acids. Zwitterionic amino acids following or attempting to follow the pathway through the membrane would, however, not be able to influence noncompetitive harmaline inhibition according to this alternate scenario. D6ves and Angelo (1996) proposed an entirely different theory to account for these complex kinetics. These authors suggested that inhibition of system y+ by cations could be attributed to an influence of the cations on the negative surface potential. That is, at low ionic strength, the negatively charged glycocalyx and possibly some phospholipid at the external surfaces of biomembranes would attract cationic amino acids and, hence, increase their concentrations near the surface. Such an effect would decrease the apparent Km value for transport, although the actual value would, of course, remain unaltered. Moreover, the effect would be masked by cations in the extracellular solution, which would, according to this theory, mimic inhibition of cationic amino acid transport. These authors also found that in the case of human erythrocytes, the effect is actually attributable to system y+L rather than to system y+. Their theory cannot, however, easily explain the much stronger inhibition by harmaline than by other cations. Moreover, in the case of system b ~ the zwitterionic amino acid taurine that is excluded as a substrate by system b ~ is, nevertheless, effective as an inhibitor of cationic amino acid transport (Fig. 4.27). To be consistent with the theory of D6ves and Angelo, taurine must, therefore, interact with the negative surface potential of cells in the same way as cations, including that both zwitterions and cations are concentrated at the surfaces of cells at low ionic strength. Zwitterionic amino acids that are also substrates of system b ~ should, therefore, also be concentrated at the membrane surface according to this theory. As discussed above, however, inhibition of system b ~ by cations is restricted to its cationic amino acid substrates; cations per se do not inhibit leucine transport by system b ~ Moreover, cations in the medium do not prevent the large effects of membrane surface potential on transport of cationic and anionic substances by CaCo-2 cells and brush border membrane vesicles (Iseki et aL, 1997). Hence, we think that it is unlikely that negative surface potential alone accounts for the complex kinetics of transport described above for systems b ~ y+, asc, and ASC.

98

4. Transport Kinetics

Molecular studies can be expected to help us to understand further the results of these kinetic studies, especially if investigators employing, say, site-directed mutagenesis, also remember to perform detailed kinetic analyses of pertinent mutant forms of transport proteins. The presence of multiple subsites to receive substrates for transport also opens the possibility that so called leaks for the migration of low-molecular-weight hydrophilic solutes may have their basis not only in slippage of normally coupled transport processes, but also in heretofore undetected transport of solutes not thought to be substrates of the process. For example, most members of the family of proteins that transport excitatory amino acids were found also to serve as channels for inorganic anions (e.g., Section XI of Chapter 3) so they presumably also have receptor sites for such anions (Zerangue and Kavanaugh, 1996a). The channels open for anion transport in the presence of excitatory anionic amino acids, which are themselves transported in a Na+dependent but C1--independent manner (Amara, 1992). In fact, some members of this family of proteins might be viewed more accurately as ligand-gated anion channels than as excitatory amino acid transporters (Sonders and Amara, 1996 and see also discussion of E A A T proteins in Chapter 6). The unappreciated channel activity of other systems and proteins thought currently to have only one transport function might yet be interpreted as leaks in instances where the basis for the mediated transport have not been identified. In the case of system b ~ the binding of cationic solutes to a subsite not required for the transport of zwitterionic amino acids may, nevertheless, lead to transport of the cationic solutes independent of whether zwitterionic amino acids are also present. By virtue of their binding to the cation receptor subsite, even cations that are not amino acids may thus be transported by system b ~ albeit with high Km values relative to the transport of amino acids. 9 While such apparently nonspecific transport of solutes by a single transport process may be relatively small and hence difficult to detect, the combined effects of such transport processes might come to require consideration for a given solute. In the cases of inorganic cations, amino acid transport systems b ~ ascl, y+, and ASC could be part of the physical basis of apparent "leaks" for these ions in biomembranes, although in our view such leaks must still involve equally complex mechanisms of transport that are characteristic 9 In fact, as discussed above, zwitterionic, cationic, and possibly even anionic amino acids should, by virtue of their positively charged amino groups, be transported by the high Km transport process postulated here. This mechanism could conceivably even account for the unanticipated high Km component of transport associated with expression of the amino acid transport related proteins BAT and CAT2a (see Section X,E below).

of all transport proteins (also see Section X,A of Chapter 3 regarding leaks for inorganic ions in biomembranes). In this regard, some Na+-dependent glutamate transporters also appear to transport several cations, including Na + in a glutamate-independent manner, and a similar result has been observed for other Na +dependent transport proteins (summarized in Vandenberg et al., 1995). Interestingly the Km value for Na + transport alone by the Na+/glucose symporter is lower than the value for its cotransport with glucose, although the velocity of Na+transport at physiological Na + concentrations is much higher for symport than for transport of Na + via this "leak" (Chen et al., 1997). Let us now turn our discussion from the sometimes complex characteristics of single transport systems and proteins to the practical problem of how the activity of a single process may be obscured by other transport and metabolic processes. In the following Section we discuss methods to minimize or deduct the effects of these other processes so that the characteristics of the transport process of interest may be examined.

X. IDENTIFICATION AND MINIMIZATION OR DEDUCTION OF PROCESSES THAT MAY OBSCURE A TRANSPORT PROCESS OF INTEREST The most frequent reason why a transport process for a solute or the solvent may be obscured or even overlooked is that the biomembrane contains several different transporters of the substrate. Transport of organic solutes may be further complicated by their metabolic alteration. Since some of the aspects of the procedure for studying transport of a given type of solute or the solvent may be specific to the substrate, and because our space is limited, we consider in this section mainly the transport of organic solutes (but see Chapter 11 for procedures for the study of the transport of other solutes). While the specific procedures for the study of transport may vary among substrate types, many of the principles for isolating and characterizing transport processes are the same regardless of the solute. One of these principles is the use of inhibitors of selected transport systems to isolate and study the activities of other transport processes. For example, ouabain can be used to inhibit Na+K+ATPase so that other transport processes for Na + and K + can be characterized separately. As we shall see, this approach also is used commonly in the study of multiple transport processes for an organic solute. In addition, it may be necessary regardless of the solute to measure transport via other processes and to deduct transport by these other processes from total transport to yield transport by the process of interest.

Identification of Obscuring Processes Each of these approaches may, however, introduce error. For example, in cases where transport that is m e a s u r e d under one condition must be deducted from transport m e a s u r e d under another, the difference is less precisely known than either of the original measurements. F u r t h e r m o r e , transport via the process of interest may be altered in ways not involving direct inhibition when experiments are p e r f o r m e d in the presence of inhibitors of alternate transport processes. We consider below several such methods for isolating the transport activity of a single process. In addition, we describe an example of how it may be reassuring to show that the characteristics of the transport process do not change when changes are applied to the experimental protocols that are used to isolate the transport activity in intact biomembranes. Since we will limit our discussion principally to organic solutes, we will begin our discussion by considering how metabolism of these solutes may influence characterization of their b i o m e m b r a n e transport.

frequently the case when accumulation of radiolabeled metabolites of the substrate occurs and can be measured. Nevertheless, investigators must remain alert to the possibility that at some time point in the uptake of an organic substrate, its metabolism may b e c o m e at least partially rate determining. If such a change does not result in a detectable decrease in the velocity of net uptake of the substrate, then errors about the characteristics of transport might be made. Such a circumstance could arise, for example, if the initial rate of accumulation of substrate owing to uptake were not easily perceived as different from the rate of accumulation of metabolites of the substrate that obtains at steady state intracellular substrate concentration (Fig. 4.28). It is also important that metabolism not become more rate determining as the external substrate concentration is

Net substrate taken up /

A. Effect of C o n c u r r e n t S u b s t r a t e M e t a b o l i s m Investigators of b i o m e m b r a n e transport must sometimes be concerned about the effects of solute metabolism on the kinetics by which a solute appears to be taken up by cells. For example, it was believed formerly that apparently saturable fatty acid transport was attributable instead to saturable metabolism of these substances and that the migration of fatty acids across biom e m b r a n e s actually was not substrate saturable. Subsequently it was shown that fatty acid transport is substrate saturable because uptake into cells could be m e a s u r e d over a time period short enough to preclude detectable fatty acid metabolism (Stremmel and Berk, 1986). Hence, saturable fatty acid metabolism cannot account for the observed saturation p h e n o m e n o n . In order for metabolism to help to limit uptake, enough substrate must accumulate inside cells to permit a significant portion of it also to be transported out of the cells via the same or other routes. W h e n the rates of uptake and exodus of a substrate reach a steady state, but net uptake of the substrate still occurs, then some other process, such as metabolism, must account for the observed net uptake of the substrate. Conversely, if metabolism is fast enough to maintain the intracellular concentration of the transported substance near zero, then such a process would actually aid in the accurate determination of kinetic parameters. Such rapid metabolism would virtually ensure the unidirectional flow of the transported substance and, hence, could help to m a k e the m e a s u r e m e n t of initial velocities relatively easy. In such cases, the metabolites must still be identifiable as substrate that has been taken up, as is

99

o

~' I--

/

/

/

~

/"

/ Substrate metabolized

~/"

Intracellular

g

0

t

2

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time ( rain )

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FIGURE 4.28 Illustration of how the characteristics of substrate metabolism might be mistaken for the characteristics of substrate transport into cells. In the schema shown, the intracellular substrate concentration ([S]) would increase initially owing to uptake with little exodus or metabolism. As the intracellular [S] increases, however, so will its exodus and metabolism. The intracellular [S] will reach a steady state when the rate of uptake is exactly equal to the rate of exodus plus metabolism. To reach an intracellular steady state IS], the initial velocity of substrate transport must exceed the rate of substrate metabolism. As the steady state [S] is approached, however, substrate exodus will become more significant and the net rate of uptake will approach the rate of metabolism. It is also assumed that metabolites of the substrate remain inside cells and are detected as representing substrate that has been taken up. In this case, the choice of a 2-min period over which to measure the initial velocity of transport would reflect both the rate of transport and the rate of metabolism. A choice of a shorter time period would, of course, reflect mainly the rate of transport, whereas a longer one would reflect mainly the rate of metabolism. In such circumstances, an inhibitor that decreases net uptake of substrate at longer but not at shorter periods of measurement probably influences substrate metabolism rather than transport. In contrast, an inhibitor of transport that is not itself transported should decrease net uptake regardless of the time period over which net uptake is measured.

| 00

4. Transport Kinetics

changed. Somewhat surprisingly, that latter phenomenon is, under some circumstances, more likely to occur as the substrate concentration is lowered rather than as it is raised (see Section X,B below). Such errors usually can be prevented if one is careful to show that, during the period over which the initial velocities are to be measured, uptake increases linearly with time across the range of extracellular substrate concentrations to be used in the study. A related practical problem arises when metabolic products of the substrate leave the cell. If formation and exodus of the product is rapid enough, then the useful metabolic "sink" described above will be lost unless the amount of product exiting the cell can be measured. This measurement would be relatively easy for some metabolic products, such as 14CO2, provided that the assay is designed to detect all such metabolic products. If the transported substrate is, however, not radiolabeled or not otherwise distinguishable from endogenous substrate, then metabolites produced specifically from transported substrate would become virtually impossible to identify. In the latter case, substrate taken up could not be distinguished from the same substance already present in the cell or produced endogenously by it. Such problems can be obviated by studying the transport of metabolism-resistant substrate analogs. While these analogs are useful in transport studies, it is still the central goal to characterize the transport of naturally occurring substrates. A principal reason for studying transport processes in the first place is to learn how solutes that are encountered by cells in situ migrate across their membranes. A final concern is that products of the transported substrate may influence substrate transport. Assuming that the products are not present initially, their effects on transport might be greater at higher exogenous substrate concentrations (depending on the Km value of the rate-limiting enzyme in the pathway producing the metabolite and the concentration of the metabolite needed to influence transport). In practice, however, such unintended influences of metabolic products appear to be absent or minimal in most studies, especially when transport is measured over time intervals short enough to reflect initial velocities. For example, if accumulation of a metabolite were to inhibit uptake, then its accumulation would result in transport that increased more slowly with time. Hence, a time interval would be chosen for subsequent studies that is short enough not to include this slowing of uptake. When such initial velocities are measured, one can calculate the initial velocity of transport for the process of interest by subtracting the initial velocities of transport via other processes from the initial velocity of total transport.

B. Deduction of Transport via A p p a r e n t l y Nonsaturable Routes Even prior to the development of modem computer software, deduction of the apparently nonsaturable component of transport depicted in Fig. 4.29 from total transport was in many instances relatively easy to accomplish. Particularly when the Km values for mediated transport are relatively low, the nonsaturable component can be so small as to be negligible at the substrate concentrations required for study of the saturable ones (e.g., Fig. 4.30). Somewhat surprisingly, however, when apparently nonsaturable transport is relatively conspicuous, it may be necessary to add a quantity to rather than subtract a quantity from total transport in order to remove the effect of the nonsaturable component of transport (Fig. 4.31). Perhaps even more surprisingly, an addition rather than a substraction is more likely to be needed at lower rather than at higher substrate concentrations (e.g., Fig. 4.31). When a saturable process is so rapid and able to accumulate substrate against its total chemical potential gradient that the intracellular total chemical potential greatly exceeds the extracellular total chemical potential after a very short time interval, it may become necessary to correct for nonsaturable transport with an addition to rather than a subtraction from measured total transport. The total chemical potential gradient formed through saturable transport would be proportionally more steep for lower substrate concentrations, since the kinetics of saturable transport are closer to first-order as the substrate concentration is reduced below the Km value. Recall also that the total chemical potential gradient of a substance that carries no net charge is proportional to the logarithm of the ratio of its intracellular to extracellular concentrations rather than to the magni-

Tt - 912 "T

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~4

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4

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[o~-Aminoisobutyrate] m M

FIGURE4.29 Deductionof nonsaturable transport from total transport yields transport owing to saturable processes. The rates of a-aminoisobutyrate (AIB) uptake into the cells of the rat diaphragm were measured over the indicated range of extracellular AIB concentrations (results of Akedo and Christensen, 1962). Reproduced, by permission, from Christensen (1975).

Identification of Obscuring Processes

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FIGURE 4 . 3 0 Nonsaturable transport of L-lysine is relatively slow at lysine concentrations needed to study transport via system b ~247 in mouse blastocysts. The extracellular concentration of L-[3H]lysine was about 1/xM at all concentrations of nonradioactive amino acids studied. Based on these data, one would determine Km and Vmax values for lysine transport by component b ~ at total concentrations of lysine of about 300/xM and below. At these concentrations, the nonsaturable component of lysine transport can be seen to be relatively small (uninhibited 1/i is marked 0.54) (adapted from Van Winkle et al., 1988a, with permission from American Society for Biochemistry & Molecular Biology).

tude of its concentration difference across the membrane. (Concentrations are again assumed to approximate activities. It is also assumed for the present illustration that the intracellular substrate concentration is initially zero.) In this case, significant nonsaturable exodus of a substrate could begin to occur while uptake of the substrate via the saturable route continues to increase almost linearly with time. It must be determined on a case by case basis whether nonsaturable transport can simply be deducted from total transport to give transport via saturable routes (e.g., Fig. 4.29) or whether more complex adjustments, such as those shown in Fig. 4.31, are needed for the particular substrate and cell type under investigation. C. Use of Inorganic I o n - D e p e n d e n c e to Deduct or Otherwise Minimize Transport of Organic Solutes by Other M e d i a t e d Processes

1. Study of Inorganic Ion-Dependent Transport Processes Numerous transport processes for organic solutes are inorganic ion dependent, and most such processes probably involve symport or antiport of the organic and

"- ~'~

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,'I

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FIGURE 4.31 The correction for passive, nonsaturable uptake may be either positive or negative depending on various factors including the extracellular total chemical potential of the substrate. The total chemical potential is proportional to the logarithm of the concentration when the net charge on the substrate is zero (a similar plot has been made for uptake of cationic and anionic substrates; Christensen and Liang, 1966a). A time interval was chosen that was short enough to approximate the initial velocity of uptake for the mediated component, but such was not the case for the nonsaturable process. At low external substrate concentrations, the cellular substrate levels achieved via the saturable component exceeded the external levels. Hence, the nonsaturable component had a net outward direction, and the total measured uptake had to be increased to reflect actual mediated uptake. At high external substrate levels, the cellular substrate concentration remained below the external level, and the net direction of nonsaturable transport was inward. In the latter case, nonsaturable uptake could be deducted from total uptake to yield mediated uptake (data form Winter and Christensen, 1964). Reproduced, by permission, from Christensen (1975).

inorganic solutes. Such symport or antiport should, of course, be demonstrated rather than assumed to occur by studying the dependence of inorganic ion transport on the presence of the organic substrate. Similarly, the stoichiometry of transport of the cosubstrates should be measured directly rather than inferred from the effect of the inorganic ion concentration on transport of the organic solute (see also Section III,B of Chapter 6). These experiments may be technically difficult to perform since the inorganic ion flux associated with organic solute transport may be small relative to the total flux of the inorganic ion. For the practical purpose of studying organic solute transport, however, the inorganic iondependence of transport of an organic solute can be exploited to study the inorganic ion-dependent process regardless of whether the inorganic ion actually is also transported. Such an approach has been used particularly frequently for the study of Na§ transport processes although it is in theory equally feasible for any inorganic ion-dependent transport process.

102

4. Transport Kinetics

For example, Na+-dependent amino acid transport has been studied in many types of cells by performing experiments in the presence and absence of Na +. The Na+-dependent component can then be obtained by deducting transport measured in the absence of Na + from transport in its presence. Deduction of the Na+-independent component also usually results in deduction of apparently nonsaturable transport, so this approach is particularly appealing when it is possible. Nevertheless, it is important to remember that when measuring initial velocity, a linear increase with time of the quantity of substrate taken up must be measured both in the presence and in the absence of the inorganic ion. While investigators usually remember the latter requirement, other important considerations are more frequently overlooked. First, measurement of the inorganic ion-dependent and -independent components of transport usually requires that a control osmolyte be substituted for the inorganic ion. It must be shown, however, that the control osmolyte does not itself influence transport. It is perhaps obvious that if Li + can substitute even in a limited way for Na + in stimulating transport, as is the case for amino acid transport system A, then a different cation should be selected to replace Na + in order to measure transport that is not Na + stimulated. It is, however, less frequently appreciated that the osmolyte that is used as a substitute may inhibit either the iondependent transport of interest (perhaps an advantage) or an ion-independent component of transport. The latter phenomenon could produce misleading results because some portion of the component of transport assumed to be ion dependent would, of course, actually be ion-independent. For example, both the Na+-dependent amino acid transport system B ~ and the Na+-independent system b ~ in mouse blastocysts are inhibited by choline (Van Winkle et aL, 1988a,b), and choline is frequently substituted as an osmolyte for Na +. Hence the difference between amino acid uptake by blastocysts in Na +- vs choline-based media would be greater than uptake by Na+-dependent systems and would represent transport via the Na+-dependent systems plus a portion of system b ~ Actually, inhibition of system b ~ extends beyond choline to other cations including Na + (see Section IX,D,5 above). The inhibition by choline is, however, greater than that by most other cations, and, unlike that by most other cations, it extends to the transport of zwitterionic as well as cationic amino acids (Van Winkle et al., 1988b, 1990c). Fortunately, other cations, such as Li +, that are used as osmolites to replace Na + have the same effect as Na + on amino acid transport system b ~ but not system B ~ in blastocysts. These other cations can therefore be used instead of choline to replace Na + in order to study Na+-dependent trans-

port by deducting transport in Na+-free medium from transport in the presence of Na +. In a similar vein, one must remain aware that the inorganic ion upon which transport is dependent may itself influence apparently ion-independent transport in unanticipated ways. In the example just discussed, Na + not only stimulates transport via system B ~ but it also surprisingly inhibits transport of cationic amino acids via system b ~ (Van Winkle et. al., 1990c). For this reason, if only sucrose or mannitol had been tested as an osmolyte to replace Na + in studies with blastocysts (or with other cells that contain systems with some of these same characteristics), then it might have been concluded erroneously that blastocysts do not contain a Na+-dependent component of cationic amino acid transport. In this case, loss of Na+-dependent system B ~ transport activity in the absence of Na + could have been obscured by an increase in transport via system b ~ (Van Winkle et al., 1985, 1990c). While conceptually simple, attempts to study inorganic ion-dependent components of transport by substituting another osmolyte for the inorganic ion may be fraught with unanticipated difficulty. One must perform enough studies to insure that the osmolyte used to replace the ion of interest does not, itself, influence transport in uncontrolled ways. The ideal replacement would be one that has all the effects on transport by various processes as the inorganic ion for which it is substituted except for the ion-dependent process that is to be studied.

2. Transport Processes That Do Not Require the Presence of an Inorganic Ion for Activity As discussed above, some transport processes for organic solutes do not appear to require the presence of an inorganic ion. In fact, these processes are sometimes inhibited by one or more ions. Fortunately, it is usually possible to identify osmolytes that are physiologically inert in regard to the transport processes intended for study, so that ion-independent transport processes may be studied in isolation from ion-dependent ones. It may also be useful to perform studies under more physiological conditions in which the inorganic ion that had been replaced for convenience of study is added back. For example, it could be hypothesized that the amino acid transport ascribed to system b ~ is simply an altered functioning of the Na+-dependent system B ~ in the absence of Na +. To test this possibility, we used a proven inhibitor of system B ~ namely BCO, that does not alter significantly transport by system b ~ If systems B ~ and b ~ were indeed separate processes, then system b ~ should be observed largely unobscured by system B ~ even in Na+-containing medium when enough BCO is present to inhibit most transport by

Identification of Obscuring Processes

system B ~ As shown in Fig. 4.32, such was observed for leucine transport in mouse blastocysts. The excess of BCO used to inhibit system B ~ also inhibited transport of leucine by a second Na+-independent system L in blastocysts. Moreover, the competitive inhibition of leucine transport by L-lysine had a Ki value that was expected for system b ~ (Fig. 4.32). Such tests of whether ion-independent transport processes are really separate from ion-dependent ones and, if they are separate entities, whether they are influenced in some unanticipated manner by the ion should be performed when the tests are possible. D. Aside from Their Fundamental Metabolic Importance, Amino Acids Are a Fortunate Choice for Study of Structural Specificity and H e t e r o g e n e i t y of Transport Mediation As indicated in the foregoing discussion, the transport of a solute or the solvent may occur via numerous routes across a biomembrane. The activities of each of these transport processes for the same substrate can be difficult to isolate for detailed characterization, especially when there is little tolerance for variability in substrate structure. Amino acid transporters may, however, each accept a wide variety of substrates albeit with different selectivities. This circumstance can be exploited for amino acid transport (and usually to a

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05 -

/

No inhibitor K~ =1301~M V.~x=36 frnol blastocyst 9 -~ min 9 -~

w=-

L~ o

I

i

5O

,

,

9

I

130

1/[ L - l e u c i n e ] , m M -1

FIGURE 4 . 3 2 Effect of 300/xM L-lysine on the Lineweaver-Burk plot for L-leucine transport via system b ~ in the presence of Na +. Ten millimolar BCO was used to inhibit the interaction of lysine and leucine with transport system B ~ and the interaction of leucine with system L. Nevertheless, the transport rates were corrected slightly for residual uptake via these other processes to produce the data shown (adapted from Van Winkle et al., 1988a, with permission from American Society for Biochemistry & Molecular Biology).

103

lesser extent for transport of other solutes) to isolate and characterize multiple transport system activities that have overlapping but different substrate specificities. Moreover, it is possible to learn about the structures of the substrate receptor sites of amino acid transport systems by examining the structures of the amino acids and the amino acid analogs that they accept or do not accept as substrates. In combination with molecular studies that are now possible for transport-related proteins (Chapters 5 to 8) it should become feasible to produce detailed descriptions of the mechanisms of protein-mediated biomembrane transport, especially in the case of amino acid transport. The molecular characterization of transport proteins for many substances began before such characterization of amino acid transport proteins. Nevertheless, it can be anticipated that the rich structural diversity of amino acids and amino acid analogs will continue to facilitate detailed characterization of the three-dimensional structures and functions of their transport-catalyzing proteins. E. Analysis of Heterogeneity of Amino Acid Transport Investigators must learn to recognize evidence that for a given instance more than one transport process may operate. They must also remember to test their proposals as to the number of mediated transport processes they anticipate are present. The number of components of transport may be exposed by examining the best statistical fit of transport data in nonlinear regression analysis or even simply by carefully inspecting data in Hofstee plots. Because of the spacing of the data points, Hofstee plots are usually more efficient at exposing multiple components of transport than are plots of other linear transformations. For example, linear transformations of transport data for the amino acid transport-related proteins BAT (related to b ~ amino acid transporter, Fig. 4.33) and CAT2a (cationic amino acid transporter 2a, Fig. 4.34) unexpectedly exposed at least two kinetically distinct components of transport. While it seems to us most likely that such expression of each of these transport-related proteins results in the expression of more than one transport activity in oocytes (see Section X,J below), it is also conceivable that one or both of these transport-related proteins alone catalyzes more than one transport process. Regardless of their origin, the presence of two or more components of transport for a given solute in the same biomembrane can obscure detection and complicate characterization of each component. Multiple components of transport for a particular substrate may be obscured both by the design of the experiments (see below) and by the nature of the transport pro-

104

4. Transport Kinetics

300

15.0

250

12.5

.c: E

200

No

10.0

\

~150

9

~

0

35

70

[Na*], mM FIGURE 6.33 Sigmoidal relationship between the Na + concentration and glycine uptake by system Gly in pigeon erythrocytes. Since a hyperbolic relationship is first-order in regard to the Na + concentration, the curves presented reflect a higher-than-first-order dependence of glycine uptake on the Na + concentration (adapted from Wheeler and Christensen, 1967 with permission from American Society for Biochemistry & Molecular Biology, Inc.).

ASC and Excitatory Amino Acid Transporters

rate transport via channels in a hyperbolic manner (e.g., Fig. 4.16 in Chapter 4) it should not be assumed that only one ion is transported at a time (see also Section III,B,2 of Chapter 7). Weaker binding of the last ion to be cotransported could conceivably account for the hyperbolic shape of such curves as discussed here for Na + transport by system ASC.

215

translocation

inner surface

*Na*

/

(4~

\.

.q ~Na* Na* , ~/j~, -.E --*S / \ Na* E E

*Na* is preferentially

unloaded inside cells

*Na§

3. A Model for Symport That Does Not Require Simultaneous Entry of all Ions or Molecules in Their Final Stoichiometric Amounts Consideration of all existing data concerning the stoichiometry of substrate cotransport by system ASC provokes modification or replacement of the model for symport developed above for this system. In the case of system ASC, the stoichiometry varies from about 4 Na + ions with each amino acid molecule in the cases of homoserine and threonine to about 4 amino acid molecules with each Na § ion in the case of proline (Thomas and Christensen, 1971; Koser and Christensen, 1971). Consequently, the model for simultaneous cotransport of Na § ions and amino acid molecules developed above must be modified to allow the stoichiometry to vary widely depending on the amino acid species. Moreover, amino acid analogs with sufficiently long and hydrophobic side-chains serve as good competitive inhibitors but not substrates for transport by some forms of system ASC. These relatively large molecules may be inherently poor substrates or they may interfere with Na § binding and consequently its apparently requisite comigration with an amino acid molecule across the membrane. It becomes somewhat difficult, however, to reconcile this concept of a well-defined substrate receptor site with the widely variable stoichiometry that must be proposed for association of different amino acid species and Na § with the site. To account for these observations Koser and Christensen (1971) in collaboration with E.L. Thomas proposed the following model in which all cotransported ions and molecules need not migrate simultaneously. In case one of this model (Fig. 6.34), the radiolabeled Na + ion, is proposed to be more likely than the radiolabeled amino acid molecule (*S) to dissociate from the system, so several labeled *Na § ions are taken up with each labeled molecule. This mechanism could account for the observed stoichiometry of homoserine or threonine cotransport with Na+. In contrast, the second case shown in Fig. 6.34 appears to apply to amino acids, such as proline, several labeled molecules of which are taken up with each labeled Na + ion (Koser and Christensen, 1971). This model needs some modification, however, to account for all of the data of Koser and Christensen (1971). In particular, the unlabeled amino acid molecule

/

9 E @

\.

S *Na§ ~ * S _ . N a . S

E/

\ S

s

*S is preferentially

unloaded inside cells

*Na*

/

(~ E

\.

S . -,,~-Na* +*S Na* E / \ ~"

E

S ~ " Na* + S

*Na* and *S are equally likely to be

unloaded inside cells

FIGURE 6 . 3 4 Scheme to account for different Na+/amino coupling ratios among amino acids cotransported with Na + by system ASC. Only the steps at the internal surface of the membrane are illustrated here. E. L. Thomas participated with B. H. Koser and H. N. Christensen in the development of this Scheme. According to this model, radiolabeled *Na + is preferentially unloaded at the inner surface of the membrane in some cases (diagram 1) while the labeled amino acid (*S) is not unloaded, whereas the reverse is true in other cases (diagram 2). Both of these phenomena as well as the third case (diagram 3) in which both substrates are unloaded (or the substrates have equal probabilities of being unloaded) would account for differences in the coupling ratio that are observed for different amino acid species (see text) (adapted from Koser and Christensen, 1971 with permission from Elsevier Science).

(S) exiting the cell in case 2 is probably of a different species than the labeled one (*S) that enters. This condition must be applied to the model in case 2 because system ASC interacts differently with the amino acid species that is in this case shown as labeled, depending on whether it is on the inside or the outside of the cell. It can be concluded that system ASC treats some amino acids, such as asparagine and proline, asymmetrically because these amino acids influence the stoichiometry of transport of other amino acids with Na + in opposite ways depending upon whether they are on the inside or the outside of the cell. When the coupling ratio (stoichiometry) for Na+:amino acid coexodus is studied in the presence of various extracellular amino acids, proline and asparagine reduce the Na+:amino acid coupling ratio of exiting substrates (Table 6.4). In contrast, intracellular proline and asparagine increase the coupling ratio for couptake of Na + and other amino acids (Table 6.5). In the latter case, however, both asparagine and proline slow the absolute rates of both Na + and amino acid entry. Consequently, although intracellular proline

216

6. Transport Proteins That Propagate Solute Gradients

TABLE 6.4 N a + / A m i n o Acid Coupling Ratios during Their Exodus from Pigeon Erythrocytes via S y s t e m ASC in Exchange for the Indicated Entering A m i n o Acid" Coupling ratio (22Na§ acid) Entering amino acid

Ala exiting

Pro Asn Ala Ser Thr

1.2-1.6 b 1.3-1.6 2.2-2.6 2.2-2.5 2.5-3.2

Thr exiting

2.7, 2.4, 3.8 3.7, 4.3,

3.2 2.9 3.7 4.3

aCoupling ratios were determined by first loading cells with 22Na+ and 3H-labeled amino acids (about 15 and 5 mM, respectively) and then measuring their efflux in medium containing unlabeled Na + (140 mM) and the indicated nonradiolabeled amino acid. The resultant initial velocities of exodus were used to calculate the coupling ratios (adapted from Koser and Christensen, 1971, with permission from Elsevier Science). bWhen a range of values is given, four determinations are included within the range.

TABLE 6.5 Effect of Loading Cells with an A m i n o Acid on the U p t a k e Rates of Na + and t h e S a m e or A n o t h e r A m i n o Acid via S y s t e m ASC in Pigeon Erythrocytes a Coupled entry b Uptake of

Cells loaded with

Experiment 1 2.5 mM Asn

Asn

2.5 mM Asn

Thr

0.5 mM Thr

Asn

0.5 mM Thr

Thr

Na §

Amino acid

Coupling ratio

54 49 57 50 191 190 244 236

64 64 87 82 34 29 78 79

0.85 0.76 0.65 0.61 5.7 6.5 3.1 3.0

and asparagine increase the coupling ratio and thus appear to favor dissociation of *Na § rather than *S (case 1 in Fig. 6.34), they must do so by somehow interacting with the transport system. Perhaps the proline and asparagine molecules are able to displace *S but they are unlikely to exit with bound *Na +. Rather, they may hold the ASC transport protein in the inward-facing conformation and hence slow the overall transport process. Only when another amino acid molecule, such as the *S that had been transported in, displaces proline or asparagine do the cosubstrates become likely to migrate back to the outside of the membrane. By then *Na + may have been displaced by an unlabeled Na + ion as in case 1, thus increasing the Na+:amino acid coupling ratio for uptake of *S. Another mechanism by which the coupling ratio may increase is through an increase in the rate at which the transporter appears to cycle. For example, hydroxyproline has a coupling ratio of about 3.2 as compared to the ratio of about 0.22 for proline (Table 6.6). Moreover, the rate of hydroxyproline transport by system ASC is about sixfold greater than the rate of proline transport (Christensen et al., 1967; Koser and Christensen, 1971; Kilberg et aL, 1981). Hence, hydroxylation of the substrate, proline, appears to increase the maximum rate at which the ASC transporter cycles by more than 80fold (6.0 • 3.2/0.22)! Part of this increased cycling may be viewed as producing an increase in the rate of amino acid uptake, but the other part appears to result in a greater Na+:amino acid coupling ratio. The reader may now recognize that the Na + exchange rate appears also to increase by more than 80-fold when hydroxyproline

TABLE 6.6

N a + / A m i n o Acid Coupling Ratios for U p t a k e via S y s t e m ASC in Pigeon Erythrocytes a Coupling ratio Na§ Mean _ SEM b

Experiment 2 0.5 mM Ser

Pro

0.5 mM Ser

Thr

0.5 mM Thr

Pro

0.5 mM Thr

Thr

177 156 368 418 296 238 484 467

44 40 240 234 19.2 18.5 117 103

4.0 4.0 1.53 1.22 15.4 12.8 4.2 4.6

alllustrative results are shown. The cells were loaded with the amino acid indicated in the second column by incubating them in a 25 mM solution of it for 4.0 to 5.25 hr. After washing the cells twice, the uptake, of 22Na+ and a 3H-labeled amino acid were measured simultaneously. The resultant initial velocities were used to calculate the coupling ratios (adapted from Koser and Christensen, 1971, with permission from Elsevier Science). bin mmol (liter of cells) -1 min -1.

Alanine Serine Threonine Proline Hydroxproline Asparagine Cysteine c

2.52 3.94 4.50 0.22 3.16 1.66 4.5

___ 0.08 +_ 0.13 +_ 0.12 +_ 0.02 ___ 0.05 ___ 0.07

acid)

Vmax(Na+)fVmax(aa) 2.41 3.74 4.66 0.25 3.13 1.84 3.4-6

aCoupling ratios in the middle column were determined as described in the footnote of Table 6.5 (adapted from Koser and Christensen, 1971, with permission from Elsevier Science). bMeans and standard deviations were calculated from numerous determinations in the original paper, and SEM was calculated here from the standard deviation reported in the original paper under the conservative assumption that only 16 determinations were made in each case. Cln the presence of 10 mM dithiothreitol.

ASC and Excitatory Amino Acid Transporters

replaces proline as the cosubstrate. This result is in contrast to the decrease in the velocity of Na + transport associated with the increase in its coupling ratio described above for the influence of intracellular asparagine and proline on the transport of other amino acids. TM A model for the Na + and amino acid receptor subsites of system ASC and similar systems is presented below (Section III,E,3). Moreover, different models to account for the observed cotransport of amino acids and Na + by several systems are presented in this and the preceding subsections. There is, however, no model that can account adequately for the observed stoichiometries in every known instance of Na+/amino acid cotransport. Hence, it is essential always to measure unknown stoichiometries of cotransport directly rather than to attempt to deduce them from a model relating transport of one cosubstrate to the concentration of another. Moreover, such measurements should be repeated for a variety of known substrates since the stoichiometry of their transport with a cosubstrate may vary as in the case of amino acid transport by system ASC.

4. The Stoichiometry of Cotransport Should Be Measured Rather Than Inferred from Cooperative Kinetic Effects Since the stoichiometry of cotransport cannot be inferred reliably from cooperative kinetic effects, it must be determined experimentally in each instance. Likewise, such models in which stoichiometry is deduced from cooperative kinetics should not be extended also to deduce the order of cosubstrate binding. Although we present a model to account for most known characteristics of EAAT-catalyzed transport in Section III,C,3 below, the model cannot be extended to other instances of co- and countertransport. Moreover, we have attempted to construct the model so that it does not imply unproven events. 19 Our success will be measured by the extent to which only new details need to be added to the model. 18 It also appears that in order to be substrates for transport by system ASC, Na + and amino acids must be separable. The positively charged side chains of cationic amino acids appear to occupy the receptor subsite for Na + (see Section III,E,3 below) but unlike Na+, the side chains are covalently linked to the rest of the amino acid molecules. Consequently, cationic amino acids are competitive inhibitors but not substrates for transport by system ASC (Thomas and Christensen, 1971) apparently owing to the inability of the positive charge on their side chains to be separated from the remainder of the molecule. 19 Technically, our model does propose the occurrence of unproven events since the transport of neither Na + nor K + by the E A A T proteins has been measured directly. Enough circumstantial evidence appears to exist, however, to warrant this conclusion, although the stoichiometry of transport remains to be determined (see Sections III,C and III,D of the text).

7.1 7

C. EAAT S y m p o r t e r s P r o p a g a t e a Na + Gradient into Gradients of Anionic A m i n o Acids As for the ASC proteins, the E A A T proteins catalyze obligatory exchange. Unlike the ASC proteins, however, E A A T proteins use the transmembrane Na + total chemical potential gradient to concentrate amino acids against their gradients. This phenomenon is possible even though exchange is obligatory because K + is by itself also a substrate. Although the total chemical potential gradient of K + is much smaller than that of Na + (Chapter 3), it lies more importantly in the reverse direction of Na+. Consequently, Na + ions usually enter the cell with an amino acid molecule, whereas K + ions are more inclined to follow their gradient out of the cell. For these reasons, both the Na + and the K + gradients drive the uptake of anionic amino acids against their total chemical potential gradients. Before we consider the kinetics of this transport in greater detail, let us review briefly the structure of E A A T proteins.

1. Structure of the E A A T Proteins As discussed above, the structures of the E A A T and ASC proteins are very similar. While the locations of the first six putative transmembrane segments seems obvious from hydropathy plots alone (Figs. 6.27 and 6.28), the locations of the more C-terminal membrane traverses are much less conspicuous in these plots. Consequently, the exact number of membrane traverses in the C-terminal region of E A A T proteins has been controversial (Fig. 6.35A vs. Fig. 6.35B), especially in regard to the long hydrophobic stretch (Fig. 6.28). In particular, a highly conserved region of this stretch (LCHS in Fig. 6.36) was proposed to cross the membrane in some models (membrane traverse number 8 in Fig. 6.35A), whereas it is placed in the cytosol in other models (Fig. 6.35B). This highly conserved sequence is interesting both because conserved regions frequently are essential to transport and because hydrophobic regions may greatly influence the free energy of binding of hydrophilic substances to the proteins. Changes in the relative free energy of their binding may help to transfer the free energy of one solute gradient into that of another. Moreover, binding of substrate by the rat EAAT2 transporter appears to alter its conformation (Grunewald and Kanner, 1995), and conformational changes seem undoubtedly to be at least part of the mechanism by which free energy conversions occur during transport (see also Chapters 3-5). Hence, the more recent finding (Wahl and Stoffel, 1996) that the LCHS in rat EAAT1 (Fig. 6.36) actually appears to traverse the membrane

2 |8

6. Transport Proteins That Propagate Solute Gradients

A

B

7

(,,.,

4

AAXFIAQ

COOH ~ FIGURE 6.35 Proposed membrane topology of E A A T proteins based primarily on their hydrophobicity plots (A and B) or on this criterion plus the results of reporter glycosylation scanning studies (C). The model of Kanai and Hediger (1992)(A) differs from that of Pines et al. (1992)(B) primarily in whether the segments labeled 7 and 8 traverse the membrane. Putative segment 7 may be too short to cross the membrane as an c~-helix, whereas putative segment 8 is in a highly conserved portion of the long hydrophobic stretch (Fig. 6.28) which is labeled LCHS in Fig. 6.36. Also shown in A and B are the locations of the aminoand carboxyl-termini, conserved SSSS and A A X F I A Q amino acid residue sequence motifs, conserved possible protein kinase A (PKA) and C (PKC) phosphorylation sites, and putative N-glycosylation sites (symbols attached to the second extracellular loop) (adapted from Kanai et aL, 1994 with permission from Federation of American Societies for Experimental Biology). An apparently more precise definition of the probable locations of the membrane traverses beyond the sixth one is shown in C for rat EAAT1. Reporter glycosylation sites were introduced into the protein or fragments of it at sites indicated by filled circles and arrows. Native N-glycosylation sites are marked with arrowheads. Subsequent detection of the locations of the glycosylation sites was used to construct the proposed topology (adapted from Wahle and Stoffel, 1996, with permission from The Rockefeller University Press and see Grunewald et aL, 1998 for another recent model for E A A T proteins).

twice (Fig. 6.35C) has considerable implications for the mechanism by which these proteins catalyze transport. Several possible regulatory phosphorylation sites are also present in the intracellular loops of all EAAT proteins (Fig. 6.35) (Gegelashvili and Schousboe, 1997). Only the conserved intracellular site in the loop between transmembrane segments 2 and 3 (Fig. 6.35) has, however, been shown in at least one case (i.e., in rat EAAT2) actually to be phosphorylated and consequently to help to regulate transport (Casado et al., 1993). Interestingly, a protein kinase C-dependent process also regulates anionic amino acid transport in cells expressing rat EAAT1. In this case, however, phosphorylation of EAAT1 occurs at a site other than the sites expected to be substrates for protein kinase C (Conradt and Stoffel, 1997). Apparently protein kinase C is needed directly or indirectly to activate phosphorylation at other sites (discussed further in Section III,A,3 of Chapter 9). In regard to the loops on the other side of the membrane, the extracellular loop between transmembrane segments 3 and 4 contains two conserved consensus se-

quences for N-linked glycosylation (Fig. 6.35). While gly+ cosylation at these sites is unnecessary for Na /glutamate symport by rat EAAT1, glycosylation is needed for formation of EAAT1 dimers in vitro (Conradt et al., 1995). If dimerization also occurs in vivo, it could lead to as yet unappreciated allosteric effects on transport. Interestingly, the rat and mouse EAAT3 proteins also have a third glycosylation site in this loop, whereas the human and rabbit EAAT3 proteins have, instead, an additional site in the first extracellular loop (Bjcr~s et al., 1996). Ongoing investigations are designed to learn how these and other structural similarities and differences among the transport proteins in the E A A T / A S C family may influence their function. Important aspects of this function include the kinetics of the transport that they catalyze. 2. Kinetics of Anionic Amino Acid Transport Careful analysis of the current induced by substrate at constant membrane electrical potential indicates that EAAT3 catalyzes the exchange of 3 Na § ions, 1 proton,

ASC and Excitatory Amino Acid Transporters

2 | 9

C

464 206

368 425

77

313

445

487,

280

342|

398~tL,,

~EL~406

q~ 495 501

273

FIGURE 6 . 3 5

(Continued)

and 1 anionic amino acid ( A A - ) molecule for 1 K + ion (Zerangue and Kavanaugh, 1996a). Since unidirectional transport of each of these solutes alone is electrogenic, the current produced by their transport together must be the sum of their individual currents. Moreover, some transport produces current in the reverse direction or is electrically silent, since K + and AA-/H+/Na + may be transported in either direction and since E A A T 3 should catalyze homo- as well as heteroexchange. 2~ When the net current resulting from the sum of these components of total transport is normalized and measured as a function of the concentrations of the various ions, the stoichiometry of transport can be deduced from the cooperativity exhibited (e.g., Fig. 6.37). In addition, K0.5 values can be calculated from the relationships between normalized current and substrate concentration (Fig. 6.37). Since K + and AA-/H+/Na + would compete for transport in the same direction, however, the observed K0.5 values 20 W e use designations, such as A A - / H + / N a + to indicate that these solutes are cotransported. Moreover, K + is by itself a substrate of the E A A T proteins. In no case do we m e a n to imply that K + may substitute for Na + or H + for cotransport with A A - .

543

514

are higher than those that would be observed for uninhibited transport. Unfortunately, the mutual cis-inhibition between AA-/H+/Na+ and K + for transport cannot be characterized by measuring the current that their transport produces. For example, the current owing to uptake of both Glu- and K + from the extracellular medium is the sum of their currents in the absence of the other (Fig. 6.38). Such would, however, be the case regardless of whether AA-/H+/Na + and K + compete for the same pathway for transport. In the case of competition, the combined current reflects both mutual inhibition of the current produced by the other substrate and the fact that the competing substrate produces current in the reverse direction. In contrast, the combined current would represent the sum of two larger individual currents in the case where K + and AA-/H+/Na + do not compete, but the sum of these currents would be the same as in the case where they do compete. Even if a scheme were used to detect a reduction in current owing to competition, however, it would not detect potentially large components of the

220

6. Transport Proteins That Propagate Solute Gradients

EAATw ~ E ,A, ,A1T 2

E A AT3

EAAT4

M , ,, , ,', ', M ': A ': S,:T,,:EMG,,A O ,, M MP': K,,Q,=V,:,E,:, VP ,, D,,:S. H,L ,G ~ . ,LOCVD. ,K. ~L ,G. .K. ,NVL H NN V ,,R M S.E~E. .PVKOH, ,R H L G L,R M G K P A R K G C PSWK R F L K NNWV M S S H G N SL F L R E S G Q R L G R V G W L Q R L Q E S L Q Q R A L R T R L RLQTMT L EHVLRF LRRNAF I .

[

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510 509 479 535

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21 The concept of effective concentration was introduced in Section II,B,5 of this chapter.

' '

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IV

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GV DL NY

ABI C R K D F A R D T G T E E--RHBKNRDVEM VY I i/K DT I D S Q H VE K EQMDVSS I E~Q ELQEAEL 561

574

FIGURE 6.36 Amino acid residue sequence alignment among members of the human EAAT subfamily of Na+-dependent amino acid transport proteins. Also shown are eight possible transmembrane segments (I to VIII) as well as the large conserved hydrophobic sequence (LCHS). If the LCHS does not traverse the membrane then it would lie outside the cell in the model presented. This extracellular location is in contrast to the membrane or cytosolic locations proposed in other models (e.g., Fig. 6.35). Possible N-linked glycosylation sites are shown in boxes (NXS or NXT) in a large extracellular loop between transmembrane segments III and IV (adapted from Arriza et al., 1997 with permission from the National Academy of Sciences, USA).

unidirectional fluxes of substrates owing to electrically silent h o m o e x c h a n g e . The contributions of h o m o e x change to the total unidirectional fluxes must be known in order to characterize accurately competition between A A - / H + / N a § and K § for transport. In this regard, since a given actual extracellular K § concentration is predicted to have a higher effective extracellular concentration at - 6 0 m V than at + 2 0 mV, the Km (and Ki) value for K + may be lower than the 17 m M determined at + 2 0 m V (Fig. 6.37). 21 For these reasons and for reasons discussed in the preceding Section (III,B), we strongly encourage investigators to charac-

I S

L I F vLLI IA TP/ C Y AG N[ , I L A SP . R MI SHYPRDEVVVKM TL. B T A IFG A.V L I T T V E HS N L S TL E K F . VI VSLAF PY.QLTYRQIK

MBS I

I

,6

N-Linked Glyco.~tLgn r;7~lLmmt~'mm.-ilmuaB-JAl~lIBnEImTImKImY R ~ K T T P V V K $ P K V A P E E A P P R R I L I Y G V Q E E . . . ING SIH V Q N F A L D L T P P P E V V Y . . . . .

1 6 8 I! ~~ ~1 B~ H~ / 1F ] ~ ~P I ~ E I ~ K B I : K IQ 1 E Y' 14 61 6 FM E E__H" 1 6 P F 219

'I L S ~ L S

S IB :

M

7 LIAI~.D. IIBI~II~BIKIRL I ~ I k - ~ B I H l i I ~ I L S G l l C J A I I ~ A I ~ E ~ I R L I ~ T ~ M / M S / I I ~ A V 2 i A ~ I ~ K L I I ~ I ~ I ~ V B A ~ S T V ~ K I ~ L / V i F C / L i l 8 S iSI]~NILII~II~NQ~L VI~N~IVI~IVK~MN$1~B~NI~A TILERMI~MI"-~IA~'~'~M VI~MI l l ~ m e 137

6,

MVPHT I LARGRDVCRRNG

terize transport by measuring unidirectional fluxes of radiolabeled substrates as well as by measuring the currents that their net transport produces. Data obtained in earlier studies of transport now known to be attributable to rat E A A T 2 (e.g., Kanner and Bendahan, 1982; Pines and Kanner, 1990) can be used also to draw conclusions similar to those above for E A A T 3 expressed in oocytes. In these earlier studies transport of radiolabeled anionic amino acids was measured in isolated m e m b r a n e vesicles and in proteoliposomes containing the purified and reconstituted transport protein. From all of these studies, one can formulate a possible m o d e l for the transport cycle of E A A T proteins.

221

ASC and Excitatory Amino Acid Transporters

A

x

~0.8

• jz~

E

.,...

"o9 0.6

N

40

.E

-20 ~ -40 -60

0.4

!,.,.,.

0

-8o ,i

0

1 := 0.8

50

,

..........

,i

i

10 100 [L-Glu] H.M ....

i

i,

i

0.4

E o 0.2 t~

Z

0

0

9

.

-6o iii! ......

. . . . . . [~1 M , 10 20 30 40 50 60 70 [K § mM

~

.

10

.

IH,]

100 nM

.

.

D

0.8 4O

~ 0.6

0,,0

o

50 100 150 200 250 300 350 [H § nM •

20

4

1SO ! .................

0

.c_

/

.~-

1 ~ 150 200 250 300 [L-Glu] ~M

E

(D N

40

.N "~ 0.4 E o 0.2 z

C

x

"o 0.6

B

= 0.8

"o9 0.6

E o 0.2

Z

1

z~

2O

0.2

Y

0

0

-. . . . . . .

25

50

75

10 _ 1 0 0 [Na'] mM , 100 125 150

[ N a § mM

FIGURE 6 . 3 7 Effects of extracellular substrate concentrations on the current induced by exchange of Glu-/H+/Na + for K + in Xenopus oocytes expressing EAAT3. Except when they were intentionally varied, external substrate concentrations were 10/zM Glu-, 32 nM H +, 60 mM Na +, and 40 mM K +. Currents owing principally to Glu-/H+/Na + uptake and K + exodus (A, B, and D) were measured at a membrane electrical potential of - 6 0 mV, whereas reverse currents (C) were measured at +20 mV. Normalized net currents (I/Imax) were fit by the least-squares method to the Equation I/Imax = [ion]n/([ion]" + Km'). According to some models, the values of n may reflect the stoichiometry of transport, and values of n greater than 1 indicate positive cooperativity. The values of n are 1.0 for Glu-, H +, and K +, and 2.3 for Na +. Apparent K0.5 values for Glu-, H +, K +, and Na + are 27 /~M, 26 nM, 17 mM, and 46 mM, respectively. The membrane electrical potentials that produce current reversal (Erev) are shown in the insets for various substrate concentrations. The relative magnitude of the slopes of the lines in the insets and the apparent stoichiometries of transport of the substrates according to this thermodynamic criterion are 1.0 Glu-, 0.79 H +, 3.17 Na +, and 0.98 K +. Both this thermodynamic method and the one involving measurement of relative cooperativity are indirect measures of stoichiometry and may be unreliable indicators of it (see text) (adapted from Zerangue and Kavanaugh, 1996a, with permission from Macmillan Magazines Ltd.). .

3. Possible Model for EAAT-Catalyzed Transport The actual fluxes of each of the co- and countersubstrates of anionic amino acid transport by E A A T proteins have not as yet been measured except as they may contribute to the net electrical current associated with transport (Fig. 6.37). Hence, the stoichiometries of transport deduced from these and other experiments need to be verified by measuring them directly (see Section III,B above). It is tempting to speculate that since the E A A T and ASC proteins are in the same family, they may have the same stoichiometries of transport. This inclination may be even more appealing since the stoichiometry deduced for Na + and anionic amino acid cotransport via E A A T proteins (e.g., Fig. 6.37) is about the same as that actually measured for transport of Na + with some amino acids via system ASC (e.g.,

alanine and hydroxyproline in Table 6.6). However, the different relationships between amino acid transport and the Na + concentration that have been observed for the E A A T (Figs. 6.37D and 6.39) and ASC (Fig. 6.30) proteins, as well as the lack of correspondence between such relationships and actual stoichiometries (e.g., Table 6.3), dissuade us from making premature judgements about the stoichiometry of transport catalyzed by E A A T proteins. Furthermore, we think that previous conclusions concerning the order of binding of Na + and anionic amino acids to E A A T proteins are premature. While existing kinetic data for these proteins and the systems that appear to contain them can be interpreted to mean that Na + binds first (e.g., Fig. 6.40), we are now reluctant to accept conclusions drawn from such studies. As discussed above, the conclusions depend on unwarranted

222

6. Transport Proteins That Propagate Solute Gradients

6~ r

-Io.

)

FIGURE 6.38 The sum of the currents produced by the simultaneous uptake of K + and Glu- (also with H + and Na +) by EAAT3 would be the same regardless of whether the substrates compete for uptake (see text for further discussion). The currents resulting from the application of 10/zM Glu-, 40 mM K +, or both of these solutes together were measured at the indicated values of the membrane electrical potential in Xenopus oocytes expressing EAAT3 ([Na +] = 60 mM) (adapted from Zerangue and Kavanaugh, 1996a, with permission from MacMillan Magazines Ltd.).

assumptions about the meaning of cooperative effects (or the lack of such effects) of one cosubstrate on the kinetics of transport of the other. Zerangue and Kavanaugh (1996a) actually used two indirect methods to estimate the stoichiometries of coand countertransport via EAAT3 (Fig. 6.37). The cooperative effects among cosubstrates described above were used to support their conclusion that 3 Na § ions are transported with each of the other co- and counter-

100

................ ' ......... ............

E

.E_ 8oX E 0

1:: 60O

/

e-

I--

---~ wr E404D

40-

1= t~ r



60 mM

5

120 mM

II/

..~

2.5mM 10.0 mM

-0.3

0

03

0.6

0.9

1 / [Glu] (mM -1)

-3

-2

-1

0

1

2

I

3

(1 / [Na*] 2 (mM-2)) x 103

FIGURE 6.40 Kinetic data consistent with the interpretation that Na + binds before Lglutamate for symport by a system in rat intestinal brush border membrane vesicles that most likely contains an EAAT protein. The Na+-dependent component of total transport is shown where the units of the initial velocity (V) are pmol (mg protein) -1 min-1. According to classic enzyme kinetic theory (e.g.; Stein, 1986), when the double-reciprocal plots for Glu- uptake at various Glu- and Na § concentrations intersect exactly on the y-axis when 1/[Glu-] is plotted (A), but to the left of this axis when 1/[Na+]2 is plotted (B), then it may be concluded that the 2 Na§ ions bind before the glutamate molecule. Furthermore, the exponents of 1 for [Glu] and 2 for [Na§ reflect their stoichiometries of transport according to kinetic theory. These and related plots may, however, be unreliable indicators of the actual stoichiometry of cotransport (see text). For this reason, such studies should not be used either to deduce the stoichiometry of transport or the order of substrate binding until the proposed kinetic model has been shown to apply to the transport process under investigation (adapted from Prezioso and Scalera, 1996, with permission from Elsevier Science).

tivity associated with B A T expression in Xenopus oocytes. The r e a d e r may recall that this transport activity resembles system b ~ (see Sections X,J and XI,E of C h a p t e r 4 and see also Section III,E below). Moreover, L-alanine and its analog, 2-amino isobutyrate (AIB), c o m p e t e for transport by this activity with about equal Km (and Ki) values (Coady et aL, 1996). These authors also found that alanine and A I B induce similar currents and amounts of L-arginine countertransport at various m e m b r a n e electrical potentials. Most importantly, in regard to the t h e r m o d y n a m i c criterion, the current reversal (i.e., equilibrium) m e m b r a n e electrical potential was the same for alanine and AIB. 23 According to these findings, the stoichiometry of exchange of arginine for either A I B or alanine appears to be about 1:1. In contrast, unidirectional alanine and A I B transport indicated that their actual stoichiometries of exchange with arginine differ by about 30-fold (Coady et aL, 1996). Hence, the t h e r m o d y n a m i c criterion appears also to be an unreliable and incompletely u n d e r s t o o d m e a s u r e of stoichiometry, at least in this instance where adequate data appear to be available to assess it. In fact, most authors 23These data are complicated somewhat because the intracellular substrates are a mixture of amino acids. Nevertheless it seems clear that the stoichiometries of exchange that would be calculated from the substrate distribution ratios at the reversal potentials (i.e., apparently at equilibrium) would be the same for alanine and AIB.

have m e a s u r e d the stoichiometry of amino acid exchange via system b ~247 directly (e.g., A h m e d et al., 1995; Chillar6n et al., 1996), whereas indirect methods appear to be used more frequently to estimate stoichiometry when one or more of the cosubstrates is an inorganic ion. In the case of E A A T proteins, one such inorganic ion, K § probably should be viewed as a substrate in its own right, rather than as serving simply to reorient E A A T proteins to receive AA-/H+/Na § on the outside of cells. True, its physiologically most conspicuous transport may be exodus, but K + undoubtedly influences the function of the proteins from outside as well as inside the cell (e.g., see Section IV,C below). Moreover, it undoubtedly competes with the cosubstrates for transport as if it were A A - / H + / N a § although this competition has not been characterized (Section III,C,2 above). The reader is also reminded that since the proposed A A - - d e p e n d e n t fluxes of Na § and K + have not actually been measured, it is conceivable (although we think unlikely) that neither of these cations actually is transported. For these reasons, we present the conservative model for E A A T - c a t a l y z e d transport shown in Fig. 6.41 as one that still needs to be verified and then to be amplified through further investigation. In the model, it is proposed that A A - and Na § are cotransported in exchange for the same substrates or K + on the other side of the m e m b r a n e . Similarly, we

224

6. Transport Proteins That Propagate Solute Gradients

kI ~ k2 ? n A ~

T~~

AAhT ~rnNa

ke~kT?

tuNa+ AAhT

+

xK~_:Na::T

k~

nAA"

TRANSLOCATION

"- NatAnT

xK

J

TRANSLOCATION

k2

Na~/!~ T .., nAA"

.

-- Nal~1A/~ T nAA"

Na~lIT

Na~IT

OUTER

INNER

FIGURE 6.41 Model for transport by E A A T proteins. These transporters (T) appear to catalyze obligatory exchange, but because of the total chemical potential gradients of Na + and K + across the plasma membrane, the system usually functions for net exodus of K + and

net uptake of the cosubstrates, Na + and anionic amino acids (AA-). Only four of the many possible rate constants are shown in order to indicate that it is not yet known whether the binding or unbinding of the cosubstrates, Na + and AA-, is ordered or random. Similarly, four of the complexes, Na+m AAn- T, are shown in the figure for convenience of illustration and are not meant to imply that the complexes formed through different orders of substrate binding are different. It is also likely that protons are cosubstrates with Na + and AA-, but their transport is not shown because the kinetics of their transport and its relationship to the transport of Na + and AA- have not been studied as extensively as the other co- and countersubstrates. The Na + and K + have also not been shown actually to migrate across the membrane, although much circumstantial evidence indicates that they are transported. Determination of the stoichiometry of their transport depends, however, on direct measurement of their migration with AA-, so the stoichiometry of transport is not indicated in the model. The modified ping-pong model shown for obligatory exchange also accounts for the trans-stimulation and probably cis-inhibition that occur between and among K + and different anionic amino acids (AA-), the later always in association with Na +.

p r o p o s e that K + is t r a n s p o r t e d by E A A T p r o t e i n s by b o t h h o m o - or h e t e r o e x c h a n g e . C o n s e q u e n t l y , these substances exhibit cis inhibition and trans stimulation as e x p e c t e d for the modified ping-pong m o d e l shown. A two-site s i m u l t a n e o u s m o d e l also is consistent with m o s t available data for this e x c h a n g e and might be m o r e likely than a ping-pong m o d e l if E A A T p r o t e i n s f o r m dimers in vivo (recall Section II,B above c o n c e r n i n g the t r a n s p o r t catalyzed by A E 1 dimers). It should also be e m p h a s i z e d that, as far as we know, each step in this m o d e l is readily reversible (Fig. 6.41). C o n s e q u e n t l y , a l t h o u g h a m i n o acid e x c h a n g e r e q u i r e s N a +, it does not a p p e a r to m a t t e r on which side of the m e m b r a n e N a + is supplied ( K a v a n a u g h et al., 1997). A p p a r e n t l y anionic a m i n o acids m a y dissociate f r o m and t h e n reassociate with the t r a n s p o r t p r o t e i n while one or m o r e N a + ions r e m a i n b o u n d to it, a l t h o u g h it has not b e e n d e t e r m i n e d w h e t h e r N a + ions can u n d e r g o

a similar process while one or m o r e anionic a m i n o acid m o l e c u l e s r e m a i n bound. As discussed above, h o w e v e r , T h o m a s and C h r i s t e n s e n (1971) c o n c l u d e d that e i t h e r a N a § ion or a zwitterionic a m i n o acid m o l e c u l e could dissociate f r o m and t h e n r e a s s o c i a t e with system A S C for t r a n s p o r t without c o n c o m i t a n t dissociation of the c o s u b s t r a t e (Fig. 6.34). H e n c e , it is r e a s o n a b l e to suppose that the s a m e m a y be true for the A S C - r e l a t e d , E A A T proteins (Fig. 6.41). F u r t h e r studies are n e e d e d b o t h to show t h a t N a § and K § ions actually m i g r a t e across the m e m b r a n e via E A A T p r o t e i n s and to d e t e r m i n e the s t o i c h i o m e t r y of their co- and c o u n t e r t r a n s p o r t with anionic a m i n o acids. It also n e e d s to be d e t e r m i n e d w h e t h e r the a p p a r e n t l y multiple N a § ions bind with equal affinity and are transp o r t e d t o g e t h e r , since such a p p e a r s n o t to be the case for the r e l a t e d A S C t r a n s p o r t e r s (Section III,B above). Similarly, the t r a n s p o r t of a p r o t o n is not shown in

ASC and Excitatory Amino Acid Transporters

the model because it is not clear in what capacity or stoichiometry it may associate with the transporter and the cosubstrates for transport. D. Use of Site-Directed M u t a g e n e s i s to Gain Insight into the M e c h a n i s m of EAAT/ ASC-Catalyzed Transport Several charged amino acid residues in hydrophobic regions of the E A A T proteins are required for normal transport activity, whereas similar mutations in several other such residues have not been found to influence transport. For example, mutation of histidyl residue 326 of the rat EAAT2 transporter (Fig. 6.28) to a lysyl, arginyl, asparagyl, or threonyl residue abolishes amino acid transport activity (Zhang et aL, 1994). Since this histidyl residue in the sixth putative membrane traverse is conserved among E A A T and ASC transporters (Fig. 6.26B) it may be required by all of them for transport. In contrast, lysyl residue 298 in the fifth putative transmembrane segment of EAAT2 does not appear to be required for transport activity (Zhang et al., 1994). Even its conversion to the very different threonyl residue reduces amino acid transport only to about 40%,and more conservative substitutions do not appear to influence transport. Since this residue is also conserved among E A A T and ASC proteins, however, it probably is needed to maintain some more subtle function of the proteins than simply whether they can transport amino acids. Similarly, a conserved aspartyl residue at the beginning of the long hydrophobic stretch (Fig. 6.28) as well as one near the end of it (i.e., D398 and D470, respectively, in the rat EAAT2) may be required for transport by all members of this family. Mutation of these residues to asparagyl, glycyl, and even glutamyl residues in rat EAAT2 abolishes amino acid transport (Pines et al., 1995). Since these two aspartyl residues are also conserved in several related bacterial anionic amino acid (Tolner et al., 1992a,b, 1995a) and even dicarboxylate (Engelke et al., 1989; Jiang et al., 1989) transporters, they may be central to the functioning of all of these proteins. In contrast, two other anionic amino acid residues near the end of the long hydrophobic stretch (E461 and D462 in rat EAAT2) do not appear to be needed for transport (Pines et al., 1995). Since all five E A A T proteins have anionic amino acid residues at these two neighboring positions (Fig. 6.36), whereas the ASC proteins and bacterial anionic amino acid transporters have only one (Fig. 6.26B and Tolner et al., 1992a,b, 1995a), it might be interesting to learn the effects of a double mutation at these two positions in the E A A T proteins. It has also not been determined whether the single anionic amino acid residue at one or the other of these positions

225

in the ASC and bacterial anionic amino acid transport proteins is needed for transport activity. Interestingly, the two related dicarboxylate transport proteins do not contain either of these anionic amino acid residues ( Jiang et aL, 1989; Engelke et al., 1989). Hence, perhaps at least one of these anionic residues is needed for recognition of the positively charged c~-amino group of amino acid substrates. On the other hand, another residue may be needed for recognition of the second organic acid group of anionic amino acids and even that of the dicarboxylate transporters. The arginyl residue at position 479 of the rat EAAT1 protein (Fig. 6.28) is conserved in mammalian and bacterial anionic amino acid transport proteins (Fig. 6.26B and Tolner et aL, 1992a,b, 1995a) and even in bacterial dicarboxylate transporters (Engelke et al., 1989; Jiang et al., 1989). This residue is, however, not conserved in the ASC proteins (Fig. 6.26B). Mutation of this arginyl residue abolishes amino acid transport by the rat EAAT1 protein (Conradt and Stoffel, 1995). We propose that the side-chain of this residue may be needed for recognition of the organic acid group on the side-chains of anionic L-amino acid substrates (or the c~-carboxylate group on D-aspartate; Gazzola et aL, 1981). In contrast, the arginyl residues at positions 122 and 280 of the rat EAAT1 protein are not as well conserved as R479 among the E A A T (Fig. 6.36) and bacterial (Engelke et al, 1989; Jiang et al., 1989; Tolner et al., 1992a,b, 1995a) transport proteins. Moreover, R122 and R280 are not needed for amino acid transport activity, although their simultaneous mutation lowers the Km value for aspartate transport by rat EAAT1 (Conradt and Stoffel, 1995). Finally, the presence or absence of two amino acid residues has particularly important implications for the physiological functions of all proteins in this family. The tyrosyl and glutamyl residues at positions 403 and 404, respectively, of the rat EAAT2 protein (Fig. 6.28) are not only required for normal transport, but their conservative mutation alters the substrate selectivity of the protein (Pines et al., 1995; Kavanaugh et aL, 1997; Zhang et al., 1998). Perhaps most importantly in regard to physiological function, the ability of the Y403F and E404D mutants to transport K + appears to be lost entirely! The presence of phenylalanyl (F) and glutaminyl (Q) residues at these positions in the ASC proteins (Fig. 6.26B) may account for their inability to countertransport K § thus rendering them also unable to use a Na § gradient to concentrate amino acids (see below). It will also be interesting to learn whether any of the bacterial proteins discussed here are able to transport K § since they also carry a glutaminyl or another uncharged residue at the position corresponding to E404, whereas tyrosyl residues at positions corresponding to Y403 are

226

6. Transport Proteins That Propagate Solute Gradients

conserved among the bacterial proteins (Jiang et al., 1989; Engelke et al., 1989; Tolner et al., 1992a,b, 1995a).

1. The Y403F and E404D Mutant Forms of Rat EAAT2 Do Not Transport K + So They Catalyze Obligatory Nonelectrogenic Anionic Amino Acid Exchange The effects on transport of the Y403F mutation are very similar to those of the E404D mutation. The major difference between the effects of the two mutations on transport is that E404D influences selectivity for anionic amino acid substrates, whereas Y403F increases the affinity of the transporter for Na+, and it somewhat broadens the ability of the transporter to receive monovalent cations in place of Na + (Pines et al., 1995; Kavanaugh et al., 1997; Zhang et al., 1998). For these reasons, we describe only the effects of the E404D mutation in detail. Both the wild-type and the E404D mutant rat EAAT2 protein transport radiolabeled D-aspartate

A EAAT2 WT i

No EAAT protein

EAAT2 E404D j

i

well, but transport by the mutant is not electrogenic (Fig. 6.42). Moreover, the normal ability of substrate to activate an anion channel in E A A T proteins is lost in the rat EAAT2 E404D mutant when K + but not DAsp-/H+/Na § is introduced into the medium of oocytes expressing the protein (Fig. 6.43). Similarly, L-Asp-/H+/ Na + but not K + stimulates exchange uptake of L-Asp-/ H+/Na+ by the E404D mutant rat EAAT2 in proteoliposomes, whereas both K + and L-Asp-/H+/Na + stimulate exchange uptake by the wild-type protein (Fig. 6.44). Finally, both AA-/H+/Na + and K + stimulate exodus of D-Asp-/H+/Na + from proteoliposomes via wild-type rat EAAT2, but K + does not stimulate exodus via the E404D mutant protein (Fig. 6.45). For these reasons, it may be concluded that the glutamyl residue at position 404 of the rat EAAT2 and probably the homologous residues of other E A A T proteins are needed for them to transport K + (Kavanaugh et al., 1997). In contrast to loss of K + transport, the E404D mutation appears to decrease the Km value for transport of both D- and Laspartate by EAAT2. D-[3H]Aspartate uptake is inhibited by the unlabeled form of this amino acid more strongly in Hela cells

i

i

40nA

' ~ [3H]D-Aspartate

0oo ~e)

A

100 l.tM D-Asp I

I ~

,

50 mM K+

I

I

I

50 s ~

f

wt

-- BCAA 9

The surface membranes of the human placenta include Na+-dependent systems A, ASC, and B ~ and Na +independent systems L and y+L for zwitterionic amino acids and additional transporters for anionic and cationic amino acids, including EAAT1 and CAT1 respectively (Moe, 1995; Malandro et al., 1996; Novak and Beveridge, 1997; Kekuda et al., 1997; Dev6s and Boyd, 1998). Transport processes with high exchange activity such as systems ASC and L may be involved in the exchange activity at the fetal face of the trophoblast; for example, extracting serine from the fetus in exchange for delivery of other amino acids. After parturition, alanine and glutamine are used by the mammary glands of lactating animals, alanine at least appearing to serve as a lipid precursor. Milk proteins are relatively enriched in Gln+Glu residues and the mammary glands are a major site of glutamine utilization during lactation (Meijer et al., 1993).

Gln Ser BCAA 9 C02

FIGURE 10.13 Amino acid exchanges between fetus and mother across the placenta. This diagram shows the major exchanges of Gly, Ser, Glu, Gin, and B C A A between the fetus and mother across the placenta. Note Gin and Gly synthesis within the placenta. See text for further details.

The fuel requirements of muscle during aerobic exercise are largely met by oxidation of glucose and free fatty acids, whereas protein is spared from this fate (Felig, 1975; Rennie, 1996, for review). Nevertheless amino acid carbon skeletons may be used for de n o v o glucose synthesis during and after exercise and a major source of these amino acids is muscle protein (Carraro et al., 1994; Rennie, 1996). There are quantitative and qualitative changes in amino acid exchange across contracting muscles during exercise and a significant net

321

Amino Acid Nutrition Under Special Circumstances

output is observed only for alanine (Felig, 1975; Carraro et al., 1994). Alanine release increases in proportion

to exercise severity and is linked to the availability of pyruvate (Carraro et al., 1994; Rennie, 1996). Nitrogen sources for alanine production from pyruvate in exercising muscle are other amino acids as well as ammonia derived from the purine-nucleotide cycle. In short-term exercise, amino-N appears to be donated from endogenous amino acids (possibly including those produced from protein breakdown), notably B C A A . A s well as pyruvate-derived alanine, alanine derived from protein breakdown also increases (from 35 to 43% of total alanine release) during low-intensity exercise, due to accelerated whole-body protein catabolism (Carraro et aL, 1994). During prolonged exercise there is net uptake of BCAA into muscle where they are oxidized as fuel with continued alanine output (Felig, 1975; Biolo et aL, 1995a). The source of these BCAA is the splanchnic bed, at least partly from breakdown of gut proteins (Williams et al., 1996). Exercise appears to stimulate amino acid transport into muscle and the stimulatory effect of exogenous amino acids on muscle protein synthesis is enhanced by prior exercise (Biolo et al., 1995a; Rennie, 1996). The mechanism of this stimulation is poorly understood but includes contribution from increased transport activity as well as increased amino acid delivery to muscle secondary to the increased blood flow (Biolo et al., 1995a; Rennie, 1996; Williams et al., 1996). Alanine released from muscle is largely extracted from plasma by the liver for gluconeogenesis, although a transient increase in plasma alanine concentration is initially observed. There appears to be an increase in hepatic alanine transport capacity during prolonged exercise, associated with increased gluconeogenesis and hepatic glucose release (Felig, 1975). During recovery from exercise, hepatic alanine uptake exceeds muscle release, and hepatic gluconeogenesis from alanine is used to enhance repletion of liver glycogen stores. Plasma glutamine is also depleted in postexercise periods and these acute effects may be cumulative if inadequate recovery periods are taken between training bouts. Athletes suffering from overtraining syndrome maintain chronically low plasma glutamine, which may have adverse effects on the gastrointestinal and immune systems (Hack et al., 1997). D. Pathophysiological Conditions Body protein stores are at risk during disease, illness, and after injury (Felig, 1975; Rennie, 1985; Waterlow, 1995; Price and Mitch, 1998, for review). The protein stores are progressively depleted and the carbon skeletons of amino acids are used for gluconeogenesis, ketogenesis, and acid-base regulation. The pathways of in-

terorgan glutamine flux therefore show marked changes in various pathological circumstances (Rennie et al., 1989; Souba, 1992; Neu et al., 1996, for review). A reduction in muscle cell volume characteristic of many pathophysiological states (H~iussinger et aL, 1993) is associated with reduced rates of muscle glutamine uptake (see Fig. 10.14), lowered muscle glutamine concentration, and net muscle catabolism (Neu et aL, 1996; Rennie et al., 1996). We have evidence for a functional link between glutamine transport and cell volume in muscle (see Section II,F), which we propose makes an important contribution to the observed increase in glutamine release from muscle in these states (Rennie et al., 1996; Low et aL, 1997a). Acquired resistance to insulin and growth hormone in peripheral tissues (mainly skeletal

A A

t.-

A

0

Od r

-5

O 1-

-10

C

o3

o3 -15

~ 9

D

/

9

t

ell) - 2 0 IO3 O i._ .i.., Z

I

200

150

250

300

Muscle cell water (ml/100g fat-free muscle dry weight)

B .i=: 7 0 0 (i)

j

D

o 600 o.

500 E

E

1

400

-o 3 0 0

E

T

-

== 200

K 100 r O

0" 0

r

~

i

J

1

2

3

Cell

a

4

~

5

~

6

j

7

water space (ml/g protein)

FIGURE 10.14 (A) Correlation of muscle cell volume with nitrogen balance in humans. Skeletal muscle cell water was measured in needlebiopsy samples from quadriceps femoris by the chloride distribution method. A, healthy subjects (n = 17); B, liver tumors (n = 5); C and D, polytrauma, day 2 (C) and day 9 (D) after trauma (n = 11); E, acute necrotizing pancreatitis (n = 6)" and F, burns (n = 4). Results are expressed as means __+SEM (adapted from H~iussinger et al., 1993, with permission from Lancet Ltd.). (B) Correlation of the effects of Gin and insulin on Gin transport activity and cell volume of rat skeletal muscle cells. 3H-Labelled Gin (0.05 m M ) transport and cell volume (3H20 space) were measured over 1 or 2 min, respectively, in NaC1 medium after 30 min exposure to basic experimental medium without or with 2 m M Gin and without or with 66 nM insulin. A, control (nothing added); B, + insulin; C, + Gin; and D, insulin + Gin. Results are means ___ SEM for four preparations.

322

10. Transport and Interorgan Nutrient Flows

muscle) during severe catabolic states may also contribute to muscle wasting (Rooyackers and Nair, 1997). 1. Acidosis

Catabolic states (e.g., type I diabetes, prolonged fasting) are associated with a metabolic acidosis owing primarily to production of ketone bodies. In such situations ureagenesis is suppressed (conserving body bicarbonate pools) and glutamine becomes the major product of ammonia detoxification (see Welbourne, 1987; H~iussinger, 1990, for reviews). Regulation of the bodily hydrogen ion economy in chronic acidosis is highly dependent on flows of glutamine between organs capable of synthesizing and releasing this substrate and the kidneys, which are capable of extracting it from the plasma to produce NH+4 for direct excretion, thus conserving HCO-3 (Welbourne, 1987; Brosnan et al., 1989). Renal glutamine utilization increases markedly during chronic metabolic acidosis in association with reduced plasma glutamine concentration, an example of concentration being sacrificed to produce the necessary flow (Souba, 1992). Such changes in plasma composition (which also include decreased [HCO-3] and [Na3] and increased [NH+4]) affect substrate supply to liver and contribute to the reduction in urea synthesis and increase in glutamine synthesis by this tissue (Welbourne, 1987; H~iussinger, 1990; Meijer et al., 1990). Inhibition of amino acid uptake into liver cells during both acute and chronic metabolic acidosis also leads to a decrease in ureagenesis (Boon et al., 1996). A major advantage of glutaminogenesis over ureagenesis during acidosis is that no bicarbonate is consumed by the glutamine synthetase reaction and indeed subsequent glutamine oxidation generates bicarbonate (Welbourne, 1987; Brosnan et al., 1989; H~iussinger, 1990). Glutamine synthesis also conserves the amino nitrogen of the ammonia which otherwise would be excreted as urea. Furthermore, the diversion of hepatic ammonia detoxification from urea synthesis to the glutamine synthetase pathway in perivenous hepatocytes facilitates renal disposition of excess NH4 +. The drawback of glutamine synthesis is that the glutamate cosubstrate ultimately comes from body protein and as wasting of muscle or other lean tissue (e.g., gut) continues (Souba, 1992; Neu et al., 1996). The combined decrease in plasma pH and glutamine concentration during chronic metabolic acidosis also helps to redirect this amino acid from the intestine (which exhibits load- and pH-dependent glutamine uptake) to the kidneys (Welbourne, 1987). Renal (but not hepatic) glutaminase is inhibited competitively by glutamate over the concentration ranges seen in vivo (Curthoys and Watford, 1995). Renal glutamate concentration falls during chronic metabolic acidosis, thereby

reducing its inhibitory effect on glutaminase, allowing increased flux through the enzyme and higher rates of glutamine breakdown (Carter and Welbourne, 1997). These observations form the basis of a model of metabolic regulation, proposed by Welbourne and colleagues (Carter and Welbourne, 1997; Welbourne, personal communication 1998) to help explain how the kidney can increase ammonia production and excretion from glutamine during chronic metabolic acidosis. In this model, the regulatory mechanism involves a feedforward regulation of glutaminase by glutamate, related to extracellular glutamate availability and glutamate transport into kidney cells (see Fig. 10.15). There is evidence that glutaminase reacts with substrates and inhibitors from the cytosolic rather than the mitochondrial matrix compartments (Kvamme et al., 1991), opening up the possibility that renal extraction of plasma glutamate contributes to modulation of glutaminase flux by altering the size of the cytosolic glutamate pool. Basolateral y-glutamyl transferase (GGT) appears to be important for generating extracellular glutamate for renal uptake; approximately 40% of the glutamine extracted from plasma by the chronically acidotic rat kidney may enter renal cells as glutamate following extracellular glutamine hydrolysis by GGT at the basolateral membrane (Welbourne and Dass, 1988). This GGT activity is highly dependent upon the external concentrations of glutamine and bicarbonate (the latter as an activator: Mu and Welbourne, 1996) such that an approximately 50% fall in both of these concentrations, as occurs in metabolic acidosis, produces a marked limitation on the glutamate available for uptake into the cell with a concomitant reduction in the intracellular pool size.

Renal Epithelium

Blood

Gin

(

Gln ~ - - - (

Lumen

Gln

NH~ +Glu " ~

Glu + NH[

~+ rico;

NH]

Renal Vein

Urine

FIGURE 10.15 Glutamine (Gin) and glutamate (Glu) exchanges in the kidney. Gin reaches the tubule lumen by glomerular filtration. Extracellular lumenal and basolateral Gin hydrolysis is catalysed by y-glutamyl transferase (GGT) with subsequent glutamate transport into the cell. Renal glutaminase is normally suppressed by high cell [Glu]. In chronic metabolic acidosis, GGT is inhibited and cell [Glu] decreases with resultant activation of glutaminase and enhanced glutamine catabolism (adapted from Welbourne and Dass, 1988, with permission from Springer-Verlag).

Amino Acid Nutrition Under Special Circumstances

Glutamine transported into kidney cells (including filtered glutamine) is thus coupled to the activated glutaminase, yielding ammonia for tubular NH4 § excretion and bicarbonate, which is released into the plasma. Elevation of the plasma bicarbonate concentration increases GGT activity with resultant increase in cellular glutamate content to close the feedback loop by inhibiting glutaminase. In this scheme, both the extracellular and intracellular signals of metabolic acidosis (HCO3- and H § respectively) act to enhance glutamine utilization for bicarbonate regeneration to help restore normal acid-base balance. 2. Sepsis and Injury Muscle glutamine stores are released after injury or infection and are used as fuel, in repairing tissues and in mounting an immune response (Souba, 1991, 1992; Neu et aL, 1996 for review). Hepatic glutamine utilization is greatly increased during sepsis (supporting increased synthesis of nucleotides, glutathione, glucose, and urea) due to a combination of factors including increased hepatic glutamine delivery (Souba, 1991). Glutamine utilization by the immune system is also increased during sepsis (Newsholme and Parry-Billings, 1990; Neu et al., 1996). This overall increase in body glutamine utilization is supplied by increased synthesis in skeletal muscle (mostly at the expense of muscle protein) and the lungs (Rennie, 1985; Souba, 1992; Neu et aL, 1996). Lymphoid cells exhibit elevated rates of amino acid transport when they enter the proliferative cell cycle during the immune response (Segel, 1992 for review). Lymphoblastoid B cells show an increase in system L transport when passing from Go into the proliferative cycle and this persists through G1 and falls during the latter part of the S phase. Activated lymphocytes increase their amino acid transport rates reflecting increased Vmax values for systems A and ASC and, in some cases, systems y+, y+L, and L (Segel 1992; Dev6s and Boyd, 1998). The elevated amino acid transport in lectin-treated lymphocytes requires new protein synthesis and occurs in parallel with other processes of lymphocyte activation. 3. Cancer An increased rate of amino acid uptake is regarded as a general feature of tumor cells, and activation of system A is a consistent early event following cell transformation in vitro (e.g., Guidotti and Gazzola, 1992, for review). Rapidly dividing cells including those of malignant tumors use glutamine as a major fuel and a tumor may become the major organ of glutamine

323

utilization in advanced malignant disease (Souba, 1991, 1992). Here the plasma glutamine concentration becomes depleted and gut glutamine utilization is therefore reduced, whereas both hepatic and muscle glutamine output increase in a vain attempt to keep pace with glutamine utilization by the growing tumor (Neu et aL, 1996). 4. Endocrine Disorders a. Diabetes Mellitus

Insulin deficiency (Type I diabetes) or resistance (Type II diabetes) have a number of direct and indirect effects on amino acid transport and metabolism (Saltiel, 1996; Rooyackers and Nair, 1997) with an overall catabolic effect on protein turnover. The major protein anabolic effect of insulin on human skeletal muscle in vivo appears to be a decrease in protein breakdown (associated with hypoaminoacidemia) rather than an increase in protein synthesis (Rooyackers and Nair, 1997). Increased amino acid supply is therefore required for insulin to even marginally stimulate muscle protein synthesis in vivo. Insulin-stimulated transport processes (e.g., system A) are less responsive to changes in whole-body nutritional status of diabetics than healthy individuals, retarding any increase in amino acid uptake into tissues immediately after feeding (McGivan and PastorAnglada, 1994; Rooyackers and Nair, 1997). This is likely to reduce the anabolic potency of amino acids in diabetes, although there may be compensatory increases in basal transport activity during longer-term uncontrolled diabetes (e.g., Low et al., 1992). Systemic effects of poorly controlled diabetes (ketoacidosis, and elevated plasma corticosteroid concentrations) may have independent effects on amino acid transport and metabolism. b. Thyroid Disease

Thyroid hormones are essential for growth but have a profound catabolic effect on skeletal muscle during both hypo- and hyperthyroidism (Rooyackers and Nair, 1997). Thyroid hormones exert physiological (and pathophysiological) effects on tissues by altering gene transcription through interactions with regulatory elements in the cell nucleus (Schwartz et al., 1993). For example, the proportion of amino acids reabsorbed from the kidney is enhanced after T3 treatment in rat, suggestive of a direct effect of T3 on expression of amino acid transporters (Fleck, 1992). Changes in hepatic T3 turnover during altered thyroid status have been reported (Schwartz et al., 1993; De-Jong et aL, 1994). We have recently studied the possible role of hepatic T3 transport activity in mediating such changes, not least because pathophysiological modulation of this process

324

10. Transport and lnterorgan Nutrient Flows

could have important consequences for whole-body management of hyper- and hypothyroid states. We found that T3 and T4 inhibited tryptophan uptake by system T in rat liver membranes to an extent dependent upon the thyroid status of the donor rat, increasing in the order hypothyroid < euthyroid < hyperthyroid, although other kinetic parameters of tryptophan uptake and T3 binding were not influenced by thyroid status (Kemp and Taylor, 1997). We speculate that these results reflect changes in association between transporter and receptor in response to alterations in plasma thyroid hormone concentrations, enabling appropriate modulation of hepatic transport (uptake and/or release) of thyroid hormones to help limit pathophysiological changes in T3 and T4 abundance (Kemp and Taylor, 1997; Taylor et al., 1998). 5. Inborn Errors of Metabolism

The interdependence between fluxes of individual amino acids for whole-body amino acid economy is shown clearly when the concentration of a single amino acid (or a small group) is either decreased or increased in the circulation. Certain inborn errors of amino acid metabolism result in extremely high plasma levels of amino acids and their metabolites. For example, phenylketonuria (PKU; defective phenylalanine hydroxylase) results in elevated plasma concentrations of phenylalanine and its metabolites (Christensen, 1987, for review), whereas maple syrup disease (MSD; defective branched-chain keto acid dehydrogenase) results in increased plasma concentrations of BCAA (especially of leucine) and their keto acids (Chuang and Shih, 1995). These disturbances in plasma amino acid composition interfere with amino acid transport across the bloodbrain barrier and cause imbalances in neurotransmitter synthesis associated with impaired brain function. Plasma excess of aromatic or branched-chain amino acids may block amino acid efflux from tissues as well as influx by competing for exchange transport through systems having a broad-substrate range such as system L (Christensen, 1987a,b, 1990, for review). This leads to sequestration of certain amino acids (including glutamine, glycine, and alanine) within tissues such as muscle and liver with a consequent reduction in their plasma concentrations. Recent unpublished data by Dr. D. H. Morton and colleagues (Clinic for Special Children, Strasburg, Pennsylvania) demonstrates that therapy leading to correction of hyperleucinemia in a newborn MSD infant (over a 6-day period) is associated with reciprocal, restorative increases in plasma concentrations of amino acids including alanine, glutamine, lysine, glycine, and serine (e.g., Fig. 10.16). These observations may reflect a release of leucine inhibition on cellular

Leucine

.....

Alanine

3000 -

750

2000 -

500

"~ (i)

.m O

250

1000

t-

,